Genome sequence comparison of two United States live attenuated vaccines of infectious laryngotracheitis virus (ILTV)

This study was conducted to identify unique nucleotide differences in two U.S. chicken embryo origin (CEO) vaccines [LT Blen (GenBank accession: JQ083493) designated as vaccine 1; Laryngo-Vac^? (GenBank accession: JQ083494) designated as vaccine 2] of infectious laryngotracheitis virus (ILTV) genomes compared to an Australian Serva vaccine reference ILTV genome sequence [Gallid herpesvirus 1 (GaHV-1); GenBank accession number: HQ630064]. Genomes of the two vaccine ILTV strains were sequenced using Illumina Genome Analyzer 2X of 36 cycles of single-end reads. Results revealed that few nucleotide differences (23 in vaccine 1; 31 in vaccine 2) were found and indicate that the US CEO strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. The sequence differences demonstrated the spectrum of variability among vaccine strains. Only eight amino acid differences were found in ILTV proteins including UL54, UL27, UL28, UL20, UL1, ICP4, and US8 in vaccine 1. Similarly, in vaccine 2, eight amino acid differences were found in UL54, UL27, UL28, UL36, UL1, ICP4, US10, and US8. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically close to both the Serva vaccine and 63140/C/08/BR (GenBank accession: HM188407) and are distinct from the two Australian-origin CEO vaccines, SA2 (GenBank accession: JN596962) and A20 (GenBank accession: JN596963), which showed close similarity to each other. These data demonstrate the potential of high-throughput sequencing technology to yield insight into the sequence variation of different ILTV strains. This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates.     

Evaluation of the protection elicited by direct and indirect exposure to live attenuated infectious laryngotracheitis virus vaccines against a recent challenge strain from the United States

In a recent study (Oldoni & García, 2007), some field strains of infectious laryngotracheitis viruses (ILTV) were characterized as genotypically different (group VI) from ILT vaccine strains. The objective of this study was to evaluate the protection elicited by one chicken embryo origin (CEO) and one tissue culture origin (TCO) vaccine against a field isolate from group VI after direct and indirect exposure to ILTV live attenuated vaccines. In phase 1 of the experiment, non-vaccinated chickens were placed into contact with the eye drop vaccinates for a period of four weeks after vaccination. Transmission of the vaccine virus to these in-contact birds was demonstrated by real time PCR and antibody production, although the in-contact birds did not become protected against disease when subsequently challenged in phase 2 of the experiment. This emphasized the importance of uniform vaccination to obtain adequate protection, both to avoid the occurrence of susceptible chickens, and to minimize the potential for reversion to virulence of live-attenuated vaccines. In phase 2, protection against challenge with a group VI field virus was assessed four weeks after vaccination by scoring clinical signs and mortality, and quantifying weight gain. Sentinel birds were added to the groups one day after challenge to assess shedding of challenge virus, using real time PCR and virus isolation, during the period 2 to 12 days post challenge. The results showed that the CEO and TCO eye drop-vaccinated chickens were protected against challenge with the group VI virus, even though it was genetically different from the vaccine strains, and that challenge virus was not transmitted from these protected birds to the sentinels.     

Characterization of infectious laryngotracheitis virus isolates from the US by polymerase chain reaction and restriction fragment length polymorphism of multiple genome regions.

Infectious laryngotracheitis (ILT) is an acute viral respiratory disease, primarily of chickens. Economic losses attributable to ILT affect many poultry-producing areas throughout the United States (US) and the world. Despite efforts to control the disease by vaccination, prolonged epidemics of ILT remain a threat to the poultry industry. Earlier epidemiological and molecular evidence indicated that outbreaks in the US are caused by vaccine-related strains. In this study, polymerase chain reaction and restriction fragment polymorphism (PCR-RFLP) of four genome regions was utilized to characterize 25 isolates from commercial poultry and backyard flocks from the US. Combinations of PCR-RFLP patterns classified the ILT virus isolates into nine groups. Backyard flock isolates were categorized in three separate groups. The ILT virus US Department of Agriculture (USDA) reference strain and the tissue culture origin (TCO) vaccine strain were categorized into two separate groups. Twenty-two isolates from commercial poultry were categorized into four groups: one group, of six isolates, showed patterns identical to the chicken embryo origin (CEO) vaccines; a second group, of nine isolates, differed in only one pattern from the CEO vaccines; a third group, of two isolates, differed in only one pattern from the TCO vaccine; a fourth group, of five isolates, differed in six and nine patterns from the CEO and TCO vaccines, respectively. Results obtained from this study clearly demonstrated that most of the commercial poultry isolates (17 of 22 isolates) were closely related to the vaccine strains. However, isolates different to the vaccine strains were also identified in commercial poultry.     

Rapid detection and characterization from field cases of infectious laryngotracheitis virus by real-time polymerase chain reaction and restriction fragment length polymorphism.

A real-time polymerase chain reaction (PCR) assay was developed to specifically amplify infectious laryngotracheitis virus (ILTV) DNA from field samples. The 222-base-pair PCR fragment was amplified using primers located in a conserved region of the infected cell protein 4 gene that was demonstrated in this work to encompass a single nucleotide polymorphism. Subsequent restriction fragment length polymorphism (RFLP) analysis of real-time PCR amplified fragments from a range of ILTV isolates using the restriction endonuclease MspI enabled differentiation between older ILTV isolates that were prevalent in the 1960s prior to the availability of vaccine strains and more recent isolates that predominantly are identical to vaccine strains. The assay, using real-time PCR, RFLP and sequence analysis, was used to characterize two recent field cases of infectious laryngotracheitis from Northern Ireland. One of the field cases was demonstrated to be similar to older "wild-type" isolates, while the other field case was identified to have a concurrent ILTV infection of both "wild-type" and vaccinal type origin. The assay described here using real-time PCR and RFLP provides a rapid, specific method that enables detection and characterization of ILTV directly from field cases.     

Comparison of virulence and restriction endonuclease cleavage patterns of infectious laryngotracheitis viruses isolated in Korea

Isolates of infectious laryngotracheitis virus (ILTV) obtained from field disease outbreaks in Korea from 1982 to 1998 were compared by virulence testing and by examining restriction endonuclease (REN) cleavage patterns of viral DNA. Based on pathogenicity tests, eight of 11 ILTV strains were classified as virulent, because these strains caused 40 to 80% mortality in specific pathogen free chickens, while three strains were classified as low virulence because these did not cause mortality. The REN cleavage patterns of the low virulence strains were identical with those of two reference vaccine strains, which were of chicken embryo origin. However, the DNA cleavage patterns of the virulent strains differed from those of both the low virulence and the vaccine strains. Furthermore, one virulent Korean strain N87278 had REN cleavage patterns that were clearly different from other virulent strains. In the present study, ILTV strains examined could be classified into two groups (virulent and low virulence strains) by pathogenicity testing, and three groups based on their REN cleavage patterns. These results suggest that most outbreaks of infectious laryngotracheitis were not likely to be associated with vaccine strains, but some were associated with viruses indistinguishable from commercial vaccine strains. At least three genetically distinct groupings of ILTV have been involved in outbreaks of infectious laryngotracheitis in Korea.     

Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks

At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the São Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to the TCO and CEO vaccines. The two Group II isolates could not be distinguished from one of the CEO vaccines. The nucleotide and amino acid sequence analyses discriminated between the Brazilian and non-Brazilian isolates, as well as between the TCO and CEO vaccines. Sequence analysis of the TK gene enabled classification of the field isolates (Group I) as virulent and non-vaccine. This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain.     

Reverse restriction fragment length polymorphism (RRFLP): A novel technique for genotyping infectious laryngotracheitis virus (ILTV) live attenuated vaccines

A novel technique, the reverse restriction fragment length polymorphism (RRFLP) assay, was developed as a means of detecting specific informative polymorphic sites in the infectious laryngotracheitis virus (ILTV) genome. During the RRFLP procedure, DNA is digested with restriction enzymes targeting an informative polymorphic site and then used as template in a real-time polymerase chain reaction (PCR) with primers flanking the informative region. The analysis of the ΔC_t values obtained from digested and undigested template DNA provides the genotype of the DNA. In this study, the RRFLP assay was applied as a method to differentiate between the two types of infectious laryngotracheitis virus attenuated live vaccines. Sequence analysis of ILTV vaccines revealed an informative polymorphic site in the 5′-non-coding region of the infected cell protein (ICP4) gene. Unique AvaI and AlwI restriction enzyme sites were identified in the tissue culture origin and chicken embryo origin attenuated vaccines, respectively. These two informative polymorphic sites were used in a RRFLP assay to genotype rapidly and reproducibly ILTV attenuated live vaccines.     

Comparison of the replication and transmissibility of an infectious laryngotracheitis virus vaccine delivered via eye-drop or drinking-water

Live attenuated vaccines have been extensively used to control infectious laryngotracheitis (ILT). Most vaccines are registered/recommended for use via eye-drop although vaccination via drinking-water is commonly used in the field. Drinking-water vaccination has been associated with non-uniform protection. Bird-to-bird passage of chick-embryo-origin (CEO) ILT vaccines has been shown to result in reversion to virulence. The purpose of the present study was to examine the replication and transmission of a commercial CEO infectious laryngotracheitis virus (ILTV) vaccine strain following drinking-water or eye-drop inoculation. Two groups of 10 specific-pathogen-free chickens were each vaccinated with Serva ILTV vaccine strain either via eye-drop or drinking-water. Groups of four or five unvaccinated birds were placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days from vaccinated and in-contact birds to assess viral replication and transmission using quantitative polymerase chain reaction. Compared with eye-drop-vaccinated birds, drinking-water-vaccinated birds showed delayed viral replication but had detectable viral DNA for a longer period of time. Transmission to chickens exposed by contact on day 0 of the experiments was similar in both groups. Birds exposed to ILTV by contact with eye-drop vaccinated birds on days 4, 8, 12 and 16 of the experiment had detectable ILTV for up to 8 days post exposure. ILTV was not detected in chickens that were exposed by contact with drinking-water vaccinated birds on day 12 of the experiment or later. Results from this study provide valuable practical information for the use of ILT vaccine.     

Genotyping of infectious laryngotracheitis virus using allelic variations from multiple genomic regions

Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4?kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level.     

The route of inoculation dictates the replication patterns of the infectious laryngotracheitis virus (ILTV) pathogenic strain and chicken embryo origin (CEO) vaccine

Infectious laryngotracheitis virus (ILTV) has a high proclivity to replicate in the larynx and trachea of chickens causing severe lesions. There is a lack of knowledge on the ability of ILTV to replicate in other respiratory associated tissues apart from in the trachea. The objective of this study was to investigate how tissues that first encounter the virus dictate further sites of viral replication during the lytic stage of infection. Replication patterns of the pathogenic strain 63140 and the chicken embryo origin (CEO) vaccine in the conjunctiva, the Harderian gland, nasal cavity and trachea were evaluated after ocular, oral, intranasal or intratracheal inoculation of specific pathogen-free chickens. Viral replication was assessed by detection of microscopic cytolytic lesions, detection of viral antigen and viral genome load. The route of viral entry greatly influenced virus replication of both strain 63140 and CEO vaccine in the conjunctiva and trachea, while replication in the nasal cavity was not affected. In the Harderian gland, independently of the route of viral entry, microscopic lesions characteristic of lytic replication were absent, whereas viral antigen and viral genomes for either virus were detected, suggesting that the Harderian gland may be a key site of antigen uptake. Findings from this study suggest that interactions of the virus with the epithelial-lymphoid tissues of the nasal cavity, conjunctiva and the Harderian gland dictate patterns of ILTV lytic replication.     

Differential diagnosis of infectious laryngotracheitis from other avian respiratory diseases by a simplified PCR procedure

A simple polymerase chain reaction (PCR)-based procedure was developed for the detection of avian infectious laryngotracheitis virus (ILTV) in chicken trachea, chorio-allantoic membrane (CAM), infected hepatoma cells and infectious cell culture supernatant. Samples were prepared by dilution in distilled water. After boiling and low speed centrifugation, samples were used for PCR analysis with two primers without special labeling. The PCR analysis for ILT virus could be completed in less than 8 h. Standard agarose gel electrophoretic analysis of the PCR products revealed a prominent band of 300 base-pairs in samples from ILTV-infected specimens, but not from specimens containing Newcastle disease virus, infectious bronchitis virus, avian adenovirus, fowlpox virus, Pachecoz or Marek's disease virus. One single ILTV infected cell or 10 plaque forming units of ILTV could be detected with this procedure. The procedure can be used for the identification of ILTV and the differentiation of ILTV from other avian respiratory tract infectants.     

Infectious laryngotracheitis virus, an alpha herpesvirus that does not interact with cell surface heparan sulfate

Among the alpha herpesviruses studied to date, the initial stage of wild-type virus attachment involves an interaction between virally encoded structural envelope glycoproteins (predominantly glycoprotein C) and cell surface heparan sulfate proteoglycans. An analysis of the infectious laryngotracheitis virus (ILTV) glycoprotein C and glycoprotein B sequences suggested that these proteins lacked consensus heparin-binding domains. This indicated that ILTV might attach to its host cell in a heparan-independent manner, distinct from other alpha herpesviruses. The infectivity of two ILTV strains, a tissue-culture-adapted vaccine strain and a virulent field challenge strain, were found to be insensitive to the presence of exogenous heparin or chondroitin. Furthermore, infectivity was retained in chicken embryonic liver cells treated with heparinase. However, 4 degrees C attachment studies and penetration studies in the presence of citrate buffer clearly demonstrated that ILTV attaches stably to and effectively penetrates chicken embryonic liver cells. Consequently, ILTV represents an alpha herpesvirus whose initial attachment step does not involve interactions with heparan or chondroitin sulfate containing proteoglycans.     

Infectious laryngotracheitis virus in chickens

Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control.     

Genetic resistance to infectious laryngotracheitis in inbred lines of White Leghorn chickens.

The susceptibility of inbred lines of chickens to graded levels of infection with a virulent isolate (wild-strain) of infectious laryngotracheitis virus (ILTV) was investigated. An association was found between the level of resistance to ILTV and the line of inbred chickens, with chickens from the J1 inbred line being more resistant to clinical ILTV than M1 and N1 inbred chickens. The N1 chickens, however, became susceptible with higher doses of ILTV, while J1 chickens remained relatively resistant to clinical disease. Studies using other inbred lines of chicken which had the same major histocompatibility complex (MHC) antigens showed similar levels of resistance or susceptibility, suggesting a possible association of the chicken MHC with resistance or susceptibility to ILTV.     

RFLP analysis of recent Northern Ireland isolates of infectious laryngotracheitis virus: Comparison with vaccine virus and field isolates from England,Scotland and the Republic of Ireland

Restriction fragment length polymorphism (RFLP) analysis was used to assist epidemiological investigations following the recent introduction of infectious laryngotracheitis virus (ILTV) to commercial poultry flocks in Northern Ireland (NI). A 4.9 kbp PCR product of the ILTV ICP4 gene was generated from each of 16 field isolates of ILTV originating from England, Scotland, NI and the Republic of Ireland (RoI) and of the single vaccine strain currently licenced for use within the United Kingdom. With the exception of isolate PV6/94 from RoI, all field isolates generated RFLP patterns, following digestion with HaeIII, similar to that obtained using the vaccinal strain. Following MspI digestion, NI isolates were indistinguishable from the vaccinal strain and recent English isolates. However, one English and one Scottish isolate, both made prior to the introduction of vaccination, and two isolates from RoI generated a second pattern following digestion with MspI.     

Differential diagnosis of fowlpox and infectious laryngotracheitis viruses in chicken diphtheritic manifestations by mono and duplex real-time polymerase chain reaction

Infectious laryngotracheitis virus (ILTV) and fowlpox virus (FPV) cause diphtheritic lesions in chicken tracheas and can simultaneously infect the same bird. A differential molecular diagnostic test, the duplex real-time polymerase chain reaction, is now reported using ILTV and FPV vaccine viruses and clinical samples from chickens, either uninfected or naturally infected with ILTV or FPV, or with both viruses. The dual virus amplification by real-time polymerase chain reaction was demonstrated to behave similarly to monoplex amplification, in spite of the fact that the real-time exponential amplification plots of the vaccine viruses were more illustrative than those of the clinical samples.     

Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus

Infectious laryngotracheitis is an acute viral respiratory disease of chickens with a worldwide distribution. Sensitive detection of the causative herpesvirus is particularly important because it can persist in the host at a very low copy number and be transmitted to other birds. Quantification of viral genome copy number is also useful for clinical investigations and experimental studies. In the study presented here, a quantitative polymerase chain reaction (qPCR) assay was developed using SYBR Green chemistry and the viral gene UL15a to detect and quantify infectious laryngotracheitis virus (ILTV) in ILTV-inoculated chicken embryos or naturally infected birds. The specificity of the assay was confirmed using a panel of viral and bacterial pathogens of poultry. The sensitivity of the assay was compared with two conventional PCR assays, virus titration and an antigen-detecting enzyme-linked immunosorbent assay. The qPCR developed in this study was highly sensitive and specific, and has potential for quantification of ILTV in tissues from naturally and experimentally infected birds and embryos.