Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F-0 cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain 1-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain 1-2 had a sequence Motif of (112)RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain 1-2 had a 7-amino-acid extension (VEILKDGVREARSSR). This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain 1-2 confirmed its avirulent nature and its Australian origin.

Development of a rapid biological assay for determination of potency of Newcastle disease vaccine (strain I-2)

A rapid biological assay based on incubation time has been developed for determination of the potency of Newcastle disease virus strain I-2 vaccine. It is based on the observation that the interval between inoculation and the first detection of haemagglutinin (HA) depends on the titre of the vaccine inoculated. Chicken embryonated eggs were inoculated with different titres (10(9), 10(6) and 10(3) EID50/0.1 ml) of vaccine and incubated for 24 h. At hourly intervals, 5 eggs from each vaccine titre were tested for the presence of HA. The results showed that the HA activity was detected from 5, 11 and 15 h after inoculation with vaccine doses of 10(9), 10(6) and 10(3) EID50, respectively. On the basis of these results it is suggested that if there is no HA detected from 5 to 11 h after inoculation of eggs with the vaccine virus, the vaccine should not be used to vaccinate chickens as it might have an infectivity titre of less than 10(6) EID50/0.1 ml, which is equivalent to the recommended single chicken dose. It is concluded that measuring the time between inoculation of the vaccine virus and the onset of HA activity might provide an estimate of the titre of the vaccine within 24 h.

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression. (c) 2006 Elsevier B.V. All rights reserved.