Attenuation and protection efficacy of ORF C gene-deleted recombinant of infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled by the use of live-attenuated vaccines. Previously we reported the complete nucleotide sequence of the ILTV vaccine strain (TCO) and identified a nonsense mutation in the gene encoding the ORF C protein. This suggested that the ORF C protein might be associated with viral virulence. To investigate this, an ILTV recombinant with a deletion in the gene encoding ORF C was constructed using the genome of the virulent United States Department of Agriculture (USDA) challenge strain (USDAch). Compared to the parental virus, the ΔORF C recombinant replicated in chicken kidney (CK) cells with similar kinetics and generated similar titres. This demonstrated that the ORF C deletion had no deleterious effects on replication efficacy in vitro. In chickens, the recombinant induced only minor microscopic tracheal lesions when inoculated via the intra-tracheal/ocular route, while the parental strain induced moderate to severe microscopic tracheal lesions, even though virus load in the tracheas were comparable. Groups of chickens vaccinated via eye-drop with the ∆ORFC-ILTV were protected to levels comparable to those elicited by TCO vaccination. To our knowledge, this is the first report that demonstrates the suitability of ∆ORFC as a live-attenuated vaccine to prevent the losses caused by ILTV.

Comparative full genome analysis of four infectious laryngotracheitis virus (Gallid herpesvirus-1) virulent isolates from the United States.

Gallid herpesvirus-1 (GaHV-1), commonly named infectious laryngotracheitis (ILT) virus, causes the respiratory disease in chickens known as ILT. The molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood, and a comparison of genomic sequences of isolates that belong to different genotypes could help identify genes involved in virulence. Dideoxy sequencing, 454 pyrosequencing and Illumina sequencing-by-synthesis were used to determine the nucleotide sequences of four genotypes of virulent strains from GaHV-1 groups I-VI. Three hundred and twenty-five open reading frames (ORFs) were compared with those of the recently sequenced genome of the Serva vaccine strain. Only four ORFs, ORF C, U(L)37, ICP4 and U(S)2 differed in amino acid (aa) lengths among the newly sequenced genomes. Genome sequence alignments were used to identify two regions (5' terminus and the unique short/repeat short junction) that contained deletions. Seventy-eight synonymous and 118 non-synonymous amino acid substitutions were identified with the examined ORFs. Exclusive to the genome of the Serva vaccine strain, seven non-synonymous mutations were identified in the predicted translation products of the genes encoding glycoproteins gB, gE, gL and gM and three non-structural proteins U(L)28 (DNA packaging protein), U(L)5 (helicase-primase) and the immediate early protein ICP4. Furthermore, our comparative sequence analysis of published and newly sequenced GaHV-1 isolates has provided evidence placing the cleavage/packaging site (a-like sequence) within the inverted repeats instead of its placement at the 3' end of the U(L) region as annotated in the GenBank's entries NC006623 and HQ630064.

Development and validation of a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry.

In this study, the development and validation of a real-time (ReTi) PCR assay is described using a Taqman labeled probe for the detection and quantitation of infectious larygotracheitis virus (ILTV) in chickens. The ReTi ILTV assay was highly specific with a quantitation limit of 100 viral template copies per amplification reaction. In experimentally infected, birds during early acute stages of infection, an average of 6.67 log(10) viral template copies/amplification reaction were detected, while at chronic late stages of infection an average of 2.86-3.27 log(10) viral template copies/amplification reaction were detected. A total of 246 tracheal swab samples collected from natural outbreaks of the disease were tested by virus isolation and the ReTi ILTV assay. Both assays agreed in 37% of the samples tested and the ReTi ILTV assay detected approximately 3.7 times more positives samples than virus isolation. A minimum of 5 log(10) viral template copies/amplification reaction were required from a tracheal swab to render a virus isolation positive result. In conclusion, the ReTi ILTV assay was highly specific, sensitive, reproducible, and capable of reliably quantifying viral nucleic acid directly from clinical samples.

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds.

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.

Characterization of infectious laryngotracheitis virus isolates: demonstration of viral subpopulations within vaccine preparations.

Infectious laryngotracheitis (ILT) is a severe acute respiratory disease of chickens caused by ILT virus. To better understand the epidemiology of the disease, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay of the glycoprotein E gene has been developed and utilized to characterize vaccine strains and outbreak-related isolates. Enzymes EaeI and DdeI were used to differentiate the tissue culture origin (TCO) vaccine from chicken embryo origin (CEO) vaccines. Two RFLP patterns were observed with enzyme EaeI, one characteristic of the TCO vaccine and a second characteristic of all CEO vaccines. Three RFLP patterns were observed with enzyme DdeI. Patterns A and B were characterized as single patterns, whereas the type C pattern was a combination of patterns A and B. Analysis of vaccine strains showed the presence of patterns A and C. Pattern A was observed for the TCO vaccine and one CEO vaccine, whereas pattern C was observed for five of the six CEO vaccines analyzed. PCR-RFLP analysis of plaque-purified virus from pattern C CEO vaccine preparations demonstrated the presence of two populations (patterns A and B). Identification of molecularly different populations of viruses within currently used ILT vaccine is the first step to develop better molecular epidemiologic tools to track vaccine isolates in the field.

Nucleotide sequencing of 5' and 3' termini of bovine viral diarrhea virus by RNA ligation and PCR.

Genomic RNA was extracted from cytopathic (CP) bovine viral diarrhea virus (BVDV) strain NADL, CP strain 72, and noncytopathic (NCP) strain SD-1 purified by ultracentrifugation. Assuming the presence of a cap structure, de-blocking of the 5' capped end of the genomic RNA was done by treatment with tobacco acid pyrophosphatase (TAP). Following decapping, the RNA molecules were ligated using T4 RNA ligase and the ligated tandem RNA templates were then amplified by primer-directed amplification (PCR). cDNA synthesis was done using reverse transcriptase with random primers and cDNA amplification was done using a negative sense primer 231-248 and a positive sense primer 12434-12451. The nucleotide sequence of the amplified product was determined by double-stranded sequencing using the Sanger di-deoxy chain termination method and an additional 'CCCCC' nucleotide sequence was identified at the ligation site. Following dATP tailing of cDNA and amplification across the 5' terminus and nucleotide sequencing, no additional nucleotides were identified on the 5' terminus. The 5' terminus as published by Collett et al., 1988b was confirmed as previously reported. Therefore, the 3' terminus includes an additional 'CCCCC' nucleotide sequence to that previously reported. Identical results were obtained when the BVDV genomic RNA was not decapped prior to RNA ligation and amplification.

Human brain tumor xenografts in nude mice as a chemotherapy model.

Two human brain tumors which were previously established in nude mice were used to determine antitumor efficacy of various therapeutic agents. These tumors were a medulloblastoma (TE-671) and a glioma (U-251) with mass doubling times of 3.5 and 5.5 days respectively as subcutaneous implants in nude mice. Intracranial (i.c.) tumor challenge was accomplished by inoculating tissue culture-grown cells of either tumor into the right cerebral hemisphere to a depth of 3 mm. Median survival time (MST) in untreated mice with 10(5) i.c. injected TE-671 cells was approximately 30 days and 53 days in the U-251 tumor. With 2 X 10(5) U-251 tumor cells the MST was 27-31 days. Groups of mice which had been inoculated with tumor were treated with various doses and schedules of antineoplastic compounds by the i.p. route. The TE-671 tumor responded to AZQ treatment with an increase in life span (ILS) of 37% compared to untreated controls and an ILS of 30% with CCNU treatment. BCNU and PCNU were ineffective. With the U-251 tumor BCNU produced an ILS of greater than 60%, with 75% cures, greater than 112% ILS with PCNU and 49% ILS with CCNU. Neither tumor responded to procarbazine, PALA, dianhydrogalactitol, D-O-norleucine or dibromodulcitol. The U-251 tumor was treated on various schedules and doses with BCNU and found to respond well on late as well as early treatment. A new drug (rapamycin) being investigated by the NCI was found to be very effective against the U-251 tumor. This model system should prove valuable in assessing the effects of various chemotherapeutic modalities against brain tumors.