Development and application of a combined molecular and tissue culture-based approach to detect latent infectious laryngotracheitis virus (ILTV) in chickens.

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens. ILTV can establish latency and reactivate later in life, but there have been few investigations of ILTV latency. This study aimed to contribute to the methodologies available to detect latent ILTV. A nested PCR was developed which was more sensitive than three other molecular methods investigated in this study. This nested PCR was then used in conjunction with in vitro reactivation culture methods that were optimized and applied to trigeminal ganglia (TG) and tracheal samples from ILTV-vaccinated commercial layer birds (n = 30). ILTV DNA was detected by nested PCR in the upper respiratory tract (URT) or eye of 22 birds. Of the remaining 8 birds, ILTV could be detected by co-culture in TG of 5 birds, with reactivated virus mostly detected 6 days post-explant (dpe). ILTV was also detected in tracheal cultures by 6 dpe. In the ILTV-positive URT samples, the virus could be characterised as vaccine strains SA2 (n = 9) or A20 (n = 5). This study provides evidence for reactivation and shedding of vaccine ILTV in commercial layer birds. Moreover, this study produced a molecular and in-vitro culture method to detect latent viral infection.

Host Src controls gallid alpha herpesvirus 1 intercellular spread in a cellular fatty acid metabolism-dependent manner.

Viral spread is considered a promising target for antiviral therapeutics, but the associated mechanisms remain unclear for gallid alpha herpesvirus 1 (ILTV). We previously identified proto-oncogene tyrosine-protein kinase Src (Src) as a crucial host determinant of ILTV infection. The present study revealed accelerated spread of ILTV upon Src inhibition. This phenomenon was independent of either viral replication or the proliferation of infected cells and could not be compromised by neutralizing antibody. Neither extracellular vesicles nor the direct cytosol-to-cytosol connections between adjacent cells contributed to the enhanced spread of ILTV upon Src inhibition. Further genome-wide transcriptional profile analyses in combination with functional validation identified fatty acid metabolism as an essential molecular event during modulation of the intercellular spread and subsequent cytopathic effect of ILTV by Src. Overall, these data suggest that Src controls the cell-to-cell spread of ILTV in a cellular fatty acid metabolism-dependent manner, which determines the virus's cytopathic effect.

Toll-like receptor (TLR)4 signalling induces myeloid differentiation primary response gene (MYD) 88 independent pathway in avian species leading to type I interferon production and antiviral response.

Engagement of toll-like receptor (TLR)4 ligand, lipopolysaccharide (LPS) with TLR4 in mammals activates two downstream intracellular signaling routes; the myeloid differentiation primary response gene (MyD)88 dependent and independent pathways. However, existence of the later pathway leading to production of type I interferons (IFNs) in avian species has been debated due to conflicting observations. The objective of our study was to investigate whether LPS induces type I IFN production in chicken macrophages leading to antiviral response attributable to type I IFN. We found that LPS elicits type I IFN response dominated by IFN-ß production. We also found that reduction in infectious laryngotracheitis virus (ILTV) replication by LPS-mediated antiviral response is attributable to type I IFNs in addition to nitric oxide (NO). Our findings imply that LPS elicits both MyD88 dependent and independent pathways in chicken macrophages consequently eliciting anti-ILTV response attributable to production of both type I IFNs and NO.

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.

Growth kinetics and transmission potential of existing and emerging field strains of infectious laryngotracheitis virus.

Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR-restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 10(2) or 10(3) plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 10(4) PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain.

Synthesis, antimicrobial, and antiviral activities of some new 5-sulphonamido-8-hydroxyquinoline derivatives.

A series of fused pyranopyrazole and pyranoimidazole, namely 5-(3,6-diamino-4-aryl-5-carbonitrile-pyrano(2,3-c)pyrazol-2-yl)sulphonyl-8-hydroxyquinolines (5a-e), 5-(6-amino-4-aryl-5-carbonitrile-pyrano(2,3-c)pyrazol-3-yl)sulphonamido-8-hydroxyquinolines (6a-e), 5-(2-thioxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl)sulphonyl-8-hydroxyquinolines (10a-e), and 5-(2-oxo-4-aryl-5-carbonitrile-6-amino-pyrano(2,3-d)imidazol-2-yl) sulphonyl-8-hydroxyquinolines (11a-e), have been prepared via condensation of some arylidine malononitriles with 5-sulphonamido-8-hydroxyquinoline derivatives 3, 4, 8 and 9. All the synthesized compounds were screened for their antimicrobial activities, and most of the tested compounds showed potent inhibition growth activity towards Escherichia coli, Pseudomonas aeruginosa (Gramnegative bacteria). Furthermore, six selected compounds were tested for their antiviral activity against avian paramyxovirus type1 (APMV-1) and laryngotracheitis virus (LTV), and the results showed that a concentration range of 3-4 µg per mL of compounds 2, 3, and 4 showed marked viral inhibitory activity for APMV-1 of 5000 tissue culture infected dose fifty (TCID(50)) and LTV of 500 TCID(50) in Vero cell cultures based on their cytopathic effect. Chicken embryo experiments show that compounds 2, 3, and 4 possess high antiviral activity in vitro with an inhibitory concentration fifty (IC(50)) range of 3-4 µg per egg against avian APMV-1 and LTV and their toxic concentration fifty (CC(50)) of 200-300 µg per egg.

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.

Pathogenicity and growth characteristics of selected infectious laryngotracheitis virus strains from the United States.

In a recent study, several US infectious laryngotracheitis virus (ILTV) strains and field isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) into nine different genotypes. All of the commercial poultry isolates were identified within genotypes IV, V, and VI. Based on the PCR-RFLP, Group IV isolates were characterized as genetically identical to the chicken embryo origin (CEO) vaccines, Group V as genetically closely related to the CEO vaccines, and Group VI as genetically different to the vaccine strains. The objective of this study was to determine the pathogenicity and growth characteristics of six ILTV commercial poultry isolates as compared with the CEO vaccine. Two isolates representative of PCR-RFLP Groups IV, V, and VI were selected. Differences in disease severity, viral tissue distribution in chickens, and plaque formation ability in cell culture were observed among viral genotypes IV, V, and VI, and between V-A and V-B isolates. Mild respiratory clinical signs were produced by IV-A, IV-B and the CEO vaccine, while VI-A and VI-B isolates produced severe respiratory signs and severe depression, and during the peak of clinical signs both isolates were re-isolated from the conjunctiva, sinus, trachea and thymus. Similarly to Group VI isolates, V-A and V-B produced severe respiratory signs, depression, and were re-isolated from conjunctiva, sinus, and trachea; on cell culture, both isolates produced significant larger plaques than any of the other isolates analysed. Overall, differences in pathogenicity and growth characteristics were observed among genetically closely related US ILTV isolates; however, complete genomes will be necessary to identify molecular determinants linked to the pathogenic viral phenotypes.

A glycoprotein I- and glycoprotein E-deficient mutant of infectious laryngotracheitis virus exhibits impaired cell-to-cell spread in cultured cells.

In alphaherpesviruses, glycoprotein I (gI) and glycoprotein E (gE) form a heterodimer that functions in cell-to-cell spread of the virus. Generally, alphaherpesvirus mutants that lack these glycoproteins are replication competent in cell culture but show a reduced capacity for cell-to-cell spread and hence smaller plaque sizes. Infectious laryngotracheitis virus (ILTV), or Gallid herpesvirus 1, is an alphaherpesvirus that causes respiratory disease in chickens. The roles of gI and gE in ILTV have not been investigated previously. In this study, a glycoprotein I and glycoprotein E deletion mutant of ILTV (gI/gE-ve ILTV) was generated by replacing the region of the ILTV genome coding for the adjacent gI and gE genes with the gene for enhanced green fluorescent protein (eGFP). This gI/E-ve ILTV was readily propagated in cell culture in the presence of wildtype ILTV (wt ILTV). However, in the absence of wt ILTV the propagation of gI/gE-ve ILTV was severely impaired. Infection of permissive cell cultures with gI/gE-ve ILTV failed to produce plaques but single infected cells could be identified by fluorescence microscopy. This suggests that gI/gE has a more significant role in the cell-to-cell spread of ILTV in vitro than in many other alphaherpesviruses.

Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment, penetration, growth curve and direct cell-to-cell spread.

The secreted alphaherpesvirus glycoprotein G (gG) works differently from other proteins. Analysis of the role of ILTV gG in virus attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve showed that gG or its antibody had no effect on ILTV attachment and penetration and that the gG antibody reduced the virus plaque size and the one-step growth curve on chicken embryo liver (CEL) cells, but gG did not affect the virus plaque size or the one-step growth curve on CEL cells. Laser scanning confocal microscopy (LSCM) detection showed that ILTV gG is located in the perinuclear region and the membrane of the CEL cells. These results suggested that ILTV gG might contribute to direct cell-to-cell transmission.

Five unique open reading frames of infectious laryngotracheitis virus are expressed during infection but are dispensable for virus replication in cell culture.

The chicken alphaherpesvirus infectious laryngotracheitis virus (ILTV) exhibits several unique genetic features including an internal inversion of a conserved part of the unique long genome region. At one end, this inversion is preceded by a cluster of five open reading frames (ORFs) of 335-411 codons, designated ORF A to ORF E, that are not present in any other known herpesvirus genome. In this report we analysed expression of these genes and identified the corresponding viral RNA and protein products. Northern blot analyses showed 3'-coterminal transcripts of ORFs A and B, and monocistronic mRNAs of ORFs C and D. ORF E is part of a 3'-coterminal transcription unit that includes the conserved glycoprotein H and thymidine kinase genes. Monospecific antisera obtained after immunization of rabbits with bacterial fusion proteins allowed detection of the protein products of ORF A (40 kDa), ORF B (34 kDa), ORF C (38 and 30 kDa), ORF D (41 kDa) and ORF E (44 kDa) in ILTV-infected cells. For functional analyses, five virus recombinants possessing deletions within the individual ORFs and concomitant insertions of a reporter gene cassette encoding green fluorescent protein were generated. All virus mutants were replication competent in cell culture, but exhibited reduced virus titres or plaque sizes when compared to wild-type ILTV. These findings indicate that the ILTV-specific ORF A to ORF E genes might be important for virus replication in the natural host organism.

Construction of recombinant avian infectious laryngotracheitis virus expressing the beta-galactosidase gene and DNA sequencing of the insertion region.

Avian infectious laryngotracheitis virus (ILTV), a herpesvirus, is a highly contagious pathogen that causes an upper respiratory tract infection in chickens. It is one of the major problems in the poultry industry worldwide. Current vaccines are not satisfactory due to the induction of latent infection. Here we describe a system for the construction of recombinant ILTV. A 4-kbp ILTV EcoRI DNA fragment was cloned into plasmid pUC13 and sequenced. Computer prediction revealed two potential open reading frames with 216 and 259 amino acid residues, respectively. The 259-residue polypeptide was serine-rich. The beta-galactosidase (beta-gal) gene of E. coli was cloned into the XhoI/Bg/II site of this DNA fragment, integrated into the ILTV genome via homologous recombination, expressed under the control of the immediate-early cytomegalovirus promoter, and caused the formation of blue plaques in the presence of X-gal. The insertion of a foreign gene into the ILTV genome and the successful expression of the incorporated gene demonstrated the potential for the construction of attenuated recombinant ILTV vaccines and the development of ILTV as vectors for polyvalent vaccines against avian upper respiratory tract infections.

Construction of recombinant infectious laryngotracheitis virus expressing the LacZ gene of E. coli with thymidine kinase gene.

We constructed the recombinant infectious laryngotracheitis virus (ILTV), CE strain, containing the LacZ gene of E. coli in the thymidine kinase gene. The growth property of the recombinant virus was almost the same as parental CE strain in chicken embryo fibroblasts. The recombinant CE strain of ILTV could be used as a live vaccine vector.

Propagation of infectious laryngotracheitis virus in an avian liver cell line.

The susceptibility of three avian cell lines (IQ1A, LMH, and QT-35) to infection by three strains of infectious laryngotracheitis virus (ILTV) was assessed both visually and by hybridization using an ILTV glycoprotein B gene probe. In the chicken liver tumor cell line (LMH), cytopathogenicity was observed at the second passage, and plaque formation was observed at the third passage. The identity of the infectious agent was verified to be ILTV by restriction endonuclease analysis of the virus genome and subsequent Southern hybridization. In contrast to LMH cells, which were a suitable host for ILTV, the quail cell line (IQ1A) was refractory to infection by this virus. Moreover, although LMH cell-adapted ILTV could initially replicate to a limited extent in the other quail cell line (QT-35), this ability was not sustained upon continual passaging.

An avian hepatoma cell line for the cultivation of infectious laryngotracheitis virus and for the expression of foreign genes with a mammalian promoter.

Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly infectious upper respiratory tract disease in chickens. Vaccine development and basic studies on ILTV have been hampered by the lack of a cell line for the cultivation of this herpesvirus which was identified in 1930. Four different avian cell lines were tested for their suitability to propagate ILTV. Here we report the successful growth of ILTV with a chemically-induced avian hepatoma cell line, while retrovirus transformed cell lines derived from permissive primary cells, were found to be non-permissive for ILTV. After multiple passaging of ILTV in the hepatoma cells, the virus could be grown up to a titre of 1 x 10(7) EID50 per ml with a replication cycle comparable to that in primary hepatocytes. Methods of plaque assay, DNA-transfection, and expression of a reporter gene were established. The gene coding for the bacterial beta-galactosidase gene under the control of the cytomegalovirus (CMV) immediate-early promotor was transiently expressed, indicating that a mammalian herpesvirus promotor was recognized by this avian cell line. Infectious ILTV virions were produced after transfecting this cell-line with purified ILTV DNA. The results indicated that the cell line is suitable for the construction of recombinant ILTV and for the molecular biological study of this important avian pathogen.

Demonstration of sites of latency of infectious laryngotracheitis virus using the polymerase chain reaction.

Mature laying chickens were inoculated intratracheally with a field strain of infectious laryngotracheitis (ILT) virus. Tracheal swabs were collected regularly from all birds for virus culture. At various times post-inoculation, pairs of birds were killed and tissues removed for detection of virus products using conventional tissue homogenization and culture, organ culture, indirect immunofluorescence (IF) and also the polymerase chain reaction (PCR). The latter was used to detect a DNA sequence from the ILT virus thymidine kinase gene. Following inoculation the birds developed mild respiratory disease with clinical signs characteristic of ILT from 3 to 10 days post-inoculation. Trachea and turbinate tissues were virus-positive as determined by virus isolation, organ culture, IF and PCR on day 4 post-inoculation. After recovery from the acute phase, virus shedding initially ceased, then intermittent, low level shedding was recorded for five of the six remaining birds. In an attempt to locate sites of latency, pairs of birds were sampled at 31, 46 and 61 days post-inoculation. Virus was not detected in upper respiratory tract or ocular tissues by conventional techniques, or in the trigeminal, proximal and distal ganglia. All tissues were also negative by PCR, except for the trigeminal ganglia of five of the six birds. All PCR-positive birds had previously shed ILT virus intermittently between days 19 and 59 post-inoculation. As we did not detect viral DNA in any of the other tissues sampled from clinically recovered birds, we conclude that the trigeminal ganglion is the main site of latency of ILT virus.

Latency and reactivation of infectious laryngotracheitis vaccine virus.

Latency and reactivation of a commercial infectious laryngotracheitis virus vaccine were demonstrated in live chickens. Virus was re-isolated at intervals between seven and fourteen weeks post-vaccination and this may be of epizootiological significance.

Herpesviruses provide helper functions for avian adeno-associated parvovirus.

The avian herpesviruses infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as the mammalian herpesvirus pseudorabies virus (PRV) were able to provide complete helper activity for the production of infectious avian adeno-associated virus (AAAV) in chicken cells. The presence of AAAV in the infected chicken cell reduced the multiplication of HVT. ILTV or PRV, however, were not affected if used as helper viruses. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious herpesvirus by plaque assays.

Studies on a live virus vaccine against infectious laryngotracheitis of chickens. I. Biological properties of attenuated strain C7.

The biological properties of tissue-culture-modified strain C7 of infectious laryngotracheitis (ILT) virus were studied. Strain C7 formed well-defined small white pocks that were distinguishable from those formed by the original strain when it was inoculated onto the chorioallantoic membrane of embryonating chicken eggs (pock marker). Strain C7 grew in chicken kidney cell cultures and embryonating chicken eggs better than the original strain. When inoculated intratracheally into chickens, strain C7 propagated primarily in the larynx, trachea, and lung, but the virus was unrecoverable after 6 days postinoculation. No contact infection was demonstrated among chickens. These biological characteristics of strain C7 were stable through five consecutive back-passages in susceptible chickens. In chickens vaccinated ocularly at 14 days of age or older, strain C7 gave good protection against challenge with the virulent strain of ILT virus. These results indicate that strain C7 may be an adequate candidate for a live virus vaccine against ILT.

[Cultivation of various avian viruses in pheasant trachea organ cultures and chick embryos]. / Kultivirane na niakoi ptichi virusi v trakhealni organni kulturi ot fazani i kokoshi zarodishi.

Stationary tracheal organ cultures of pheasants and chick embryos, treated with a mucolytic agent were used to study the ciliostatic effect of the following viruses: fowl pest (FPV), Newcastle disease (NDV), infections laryngotracheitis (ILV), and infections bronchitis (IBV) - strain Beaudette. In chick embryo tracheal cultures ciliostasis was found to set in as follows: for FPV - at the 24th hour; for NDV - at the 72nd-120th hour; for ILV - at the 168-192nd hour; and for IBV-at the 72nd-96th hour. The ciliostatic effect produced by NDV and IBV coincided in terms of time and dynamics. In pheasant tracheal organ cultures ciliostasis set in as follows: for FPV - at the 24th hour; for NDV - at the 72nd-120th hour; and for ILV - at the 168-192nd hour. This system was shown to be unsusceptible to IBV which produced no effect whatever. In mixed organic cultures, containing several tracheal segments of pheasants and chick embryos each per petri dish IBV did bring about ciliostasis in the chick embryo segments only, within the period after inoculation as cited above. It is stated that pheasant tracheal organ cultures could be employed in the case of an express initial differentiation of FPV, NDV, and ILV, recording the time period for the setting in of full ciliostasis following the inoculation of the respective viruses. Discussed is the possibility of identifying IBV, using mixed tracheal organ cultures of pheasants and chick embryos.