Triple plaque purified strains of duck hepatitis virus and their potential as vaccines

Individual triple plaque purified strains of attenuated duck hepatitis virus can protect ducklings against virulent challenge with the virus but they are as genetically unstable as their parent vaccine strains and are transmissible by direct contact. The use of rapid passage is advocated as a method to improve these parameters.     

Inhibition of mouse hepatitis virus multiplication by an oligonucleotide complementary to the leader RNA.

An oligonucleotide complementary to a leader RNA of positive-stranded mouse hepatitis virus (MHV) was tested for the effect on the viral multiplication in mouse DBT cells. A 14-mer antisense oligonucleotide contained a sequence complementary to the conserved pentanucleotide sequence, UCUAA, of the leader RNA. A treatment of MHV-infected cells with the antisense oligonucleotide at concentrations from 5 to 25 microM had an inhibitory effect on the viral multiplication and reduced the synthesis of viral specific mRNA and proteins. No inhibitory effect was observed when the cells were treated with sense oligonucleotide and oligonucleotide which contained unrelated sequences at concentrations from 1 to 10 microM. These results showed that antisense oligonucleotide against the leader RNA reduced the multiplication of positive-stranded RNA virus, MHV.     

Thermostability of duck hepatitis virus.

The comparative thermostability of 4 duck hepatitis (DH) viruses were tested at various temperatures for different times. Titer of duckling-passaged, pathogenic DH virus decreased from 10(4.50) to 10(2.33) and 10(2.20) median infective doses (ID50/0.1 ml, respectively, in 2 tests; titer of chicken embryo-passaged, nonpathogenic, but embryo-lethal, DH virus decreased from 10(6.00) to 10(0.46) and from 10(6.62) to 10(0.63) ID50/0.1 ml, respectively; duck embryo fibroblast culture-passaged and duck embryo liver cell culture-passaged, chicken ebryo-infective, but nonlethal, DH viruses were completely inactivated or nearly so after being kept at 56 C for 30 minutes. Duckling-passaged DH virus was not detected on day 21, whereas 10(0.62) ID50 of chicken embryo-passaged DH virus per 0.1 ml remained on day 32 when being kept at 37 C. Titer of chicken embryo-passaged DH virus decreased from 10(7.00) to 10(1.16) ID50/0.1 ml after being kept at room at room temperature for 150 days, to 10(5.17) ID50/0.1 ml after being kept at 4 C for 70 weeks, to 10(6.17) ID50/0.1 ml after being kept at -20 C for 70 weeks, and to 10(6.38) ID50/0.1 ml after being kept at -60 C for 1 year.     

Immunoperoxidase plaque staining for the detection of pseudorabies virus.

A quantitative indirect immunoperoxidase plaque staining method was developed for the detection of pseudorabies virus infection in a pig kidney cell line (PK-15). The method is rapid and specific and foci of infection, represented by stained plaques, are easily counted by the unaided eye. Possible modification of this technique in a plaque reduction assay for the detection of antipseudorabies virus antibody is also discussed.     

Properties of non-neurovirulent plaque-forming mutants of Newcaslte disease virus.

The mesogenic Hertfordshire (H) strain, a vaccine strain of Newcastle disease virus, represents a heterogeneous virus population differing in physical, chemical, and biological properties. Small (S) and large (L) plaque-forming mutants were isolted from strain H in chick embryo fibroblast cell cultures. Part of the S mutants lacked neurovirulence, with an intracerebral pathogenicity index for day-old chicks of 0 to 0.46 vs 1.0 to 1.4 for L mutants. S mutants grew only to a very low titer in the brain of day-old chicks and cleared from the brain by the sixth day, whereas L mutants killed the chicks after a 1000-fold titer rise. The S mutants caused a complete cell destruction in fibroblast culture by 12 hours, but they had 10--20 times lower virus yields than the L mutants. The infective titers of S and L mutants grown in allantoic cells were identical, but the infectivity to hemagglutinin ratio was 10 to 50 times lower for S than for L. The thermostability of infectivity and hemagglutinin varied with the different mutants. As for the immunogenicity of the mutants, the minimum dose of S mutants inducing full protection by subcutaneous inoculation was 10(6) plaque-forming units per chicken, thus being about 100 times less immunogenic than either the L mutants or the parent strain H.     

Genetic and physiological characterization of temperature-sensitive mutants of bluetongue virus.

Complementation studies were carried out, using temperature-sensitive (t-s) mutants of blue-tongue virus (BTV). The results proved to be inconclusive as only low indices of complementation were obtained. No discrepancy was found between the previous classification of these mutants in 6 recombination classes and the complementation data recored. In general, the t-s mutants require a latent growth period of 16-20 h at 28 degrees C and maximum titres can be demonstrated 40-48 h post-infection. One mutant, (F211), however, consistently had a growth lag phase of 32 h. Mutants of the 6 recombination groups were further classified into 2 groups by temperature-shift studies. One calss of mutants expressed their t-s lesion prior to 24 h and the other class only after 24 h post-infection. Mutant F73 was found to be defective in its ability to synthesize ssRNA at a late stage in the replication cycle at the non-permissive temperature.     

Transmissible gastroenteritis virus: plaques and a plaque neutralization test.

A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.     

Duck virus hepatitis: serial passage of attenuated virus in ducklings.

The safety of three attenuated virus vaccines of proven efficacy against duck virus hepatitis was assessed by controlled laboratory studies which involved the serial transmission of the virus through groups of two-day-old ducklings known to be susceptible to the disease. Each vaccine was initially derived from a different source. Enhancement of virulence which resulted in deaths from the disease in test groups of ducklings occurred in each instance.