The potential contribution of scavenging chicken flocks to village populations in developing countries is being reassessed partly as the result of the development in Australia of new Newcastle disease vaccines for village use. These vaccines, based on avirulent thermostable Australian strains of Newcastle disease virus (NDV) (initially strain V4 and more recently strain I-2), can be produced locally and are not entirely reliant on a cold chain. They are finding use in many developing countries. The present study deals with some practical aspects of the production and use of I-2 vaccine in developing countries. It also addresses some fundamental aspects of I-2 virus and contributes to the international database on comparative virology.
The assay of avirulent strains of NDV has relied on the use of embryonated eggs, as these viruses are poorly cytopathogenic in cultured avian
cells. Embryonated eggs are unsuited for repetitive assays, being both time-consuming and expensive. I-2 virus was shown to produce cyctopathic changes in chicken embryo kidney cells grown in microtitre trays. End points could be determined laboriously by careful microscopic examination or readily by gross examination of fixed and stained cultures. Cell culture was less sensitive than egg inoculation in determining viral infectivity, but endpoints obtained by both methods were highly correlated. Cell culture was used later in this study when repeated titrations of the infectivity of I-2 virus were required.
Although I-2 virus is relatively thermostable, I-2 vaccines must still be kept as cool as possible. Diluents that protect against thermal inactivation could extend shelf life when storage at ambient temperatures was required. I-2 virus was suspended in several diluents that are used to protect viruses during lyophilization. Inactivation rates were
established on storage at 22-25°C. The diluents that gave the best protection were 5% lactalbumin, 5% skim milk, 1% gelatin, 1% polyvinyl pyrrolidone (PVP) and a complex mixture (SPGAE) of sucrose, phosphates, glutamic acid, bovine serum albumin and EDTA.
In some circumstances, I-2 vaccine will be produced in liquid form in provincial laboratories and used within a fortnight. There will be no time for conventional quality assurance tests. It would be advantageous to develop a method for rapid estimation of potency that does not involve titration in eggs, a process requiring at least 4 days. Embryonated eggs were inoculated with varying doses of I-2 virus, and the intervals until progeny virus could be detected by a haemagglutination test were determined. A standard chicken dose of I-2 vaccine (106 50% embryo infectious doses) yielded detectable haemagglutinin 11 hours after inoculation. This simple overnight assay may suffice
to indicate the potency of a newly harvested vaccine.
Although I-2 vaccine is usually administered by eye drop in the field, many administrators would prefer a food-based vaccine. White (polished) rice is an obvious choice, being readily available in many countries, and acceptable to chickens. However raw white rice rapidly destroys the vaccine virus that is applied to it. Boiling the rice results in an excellent vehicle for the vaccine, but one that is rapidly subject to bacterial spoilage. Raw white rice was mixed with vegetable oil and then coated with I-2 vaccine and stored at 22-25°C. Recovery of vaccine virus from oiled rice was only slightly less than recovery from boiled rice after 0.5 or 24 hours. Oiled rice may be a suitable vehicle for a food-based I-2 vaccine.
Unselected strain I-2 virus possessed a degree of thermostability which was enhanced by heat selection when the I-2 master seed was produced. At present
the production of I-2 vaccine involves two passages in eggs (to working seed and to vaccine) without further heat selection. Thermostability is retained. Vaccine production could be increased if further passages could be interposed between master seed and vaccine without loss of thermostability. I-2 vaccine was serially passaged 20 times in embryonated eggs and thermostability of infectivity at 56 °C was determined after each 5 passages. Thermostability was virtually unaltered after 5 passages, rate constants (k x 10-3/minutes-1) being 3.6 at passage 1 and 3.3 at passage 5. Thermostability declined with further passage. Haemagglutinating activity declined less rapidly.
V4, an avirulent, enteric strain of NDV was isolated in 1966. Strain I-2 was isolated in the early 1990s and was a representative of viruses that were believed to be evolving tropism for the respiratory tract. Later isolates of NDV in Australia included a pathogenic strain, and a
progenitor that formed a link between the pathogenic and the apathogenic isolates. It was important to define the biological and molecular characteristics of strain 1-2 to differentiate it from these other viruses.
A reverse transcription polymerase chain reaction (RT-PCR) was developed that allowed strains V4 and I-2 to be detected in chicken tissues, and that could distinguish between these viruses. Chickens were vaccinated with either strain I-2 or strain V4 and various tissues were sampled for virus over the next 7 days. Both viruses were detected in samples from both the respiratory and digestive tracts. Strain I-2 was detected earlier than V4 in the respiratory tract and persisted for longer. Strain V4 was detected earlier than strain I-2 in the digestive tract, and persisted for longer. It was concluded that the two viruses exhibited different tissue tropisms.
Amino acid sequences, deduced from nucleotide sequences, are now
widely used as indicators of the pathotype of strains of NDV and in tracing the evolutionary history of isolates. The cleavage site of the fusion protein and the carboxyl terminus of the haemagglutinin-neuraminidase protein have been useful sites. Molecular studies had not previously been undertaken with strain I-2. Single tube RT-PCR techniques were developed for use with I-2 virus, the amplicons were sequenced and the amino acid sequences deduced.
The cleavage activation site of I-2 was 112RKQGR116 differing only in the substitution of R for G at residue 112 from the motif of V4 and other avirulent strains of NDV. At the N terminus of the F1 protein, I-2 shared with V4 and other avirulent strains the motif 117LIG119 and not the 117FIG119 motif of virulent strains.
carboxyl terminus of the haemagglutinin-neuraminidase protein, strain I-2 had a 7 amino acid extension. This indicates a different lineage from V4 and similar avirulent strains (45 amino acid extensions) and Australian virulent strains and their apparent precursors (9 amino acid extensions).