Publications

To assess the effect of various vaccine strains on replication and shedding of virulent Marek's disease virus from experimentally infected chickens, quantitative PCR (q-PCR) methods were developed to accurately quantify viral DNA in infected chickens and in the environment in which they were housed. Four groups of 10 chickens, kept in poultry isolators, were vaccinated at 1 day old with one of four vaccines covering each of the three vaccine serotypes, then challenged with very virulent MDV strain Md5 at 8 days of age. At regular time-points, feather tips were collected from each chicken and poultry dust was collected from the air-extract prefilter of each isolator. DNA was extracted from feather and dust samples and subjected to real-time q-PCR, targeting the US2 gene of MDV-1, in order to measure Md5 level per 104 feather tip cells or per microgram of dust. Accuracy of DNA extraction from dust and real-time q-PCR were validated by comparing either q-PCR cycle threshold values or the calculated MDV genome level; for use in q-PCR, DNA was extracted from serial dilutions of MDV-infected dust diluted with noninfected dust, or DNA from MDV-infected dust was diluted with DNA from noninfected dust. The results confirmed the accuracy and sensitivity of dust DNA extraction and subsequent q-PCR and showed that differences in virus levels between dust samples truly reflect differences in shedding. Vaccination delayed both replication of Md5 in feather tips and shedding of Md5. First detection of Md5 in feather tips always preceded or coincided with first detection in dust in each group. pCVI988 and HVT+SB-1 were the most efficient vaccines in reducing both replication and shedding of Md5. There was close correlation between mean virus level in feathers of each group and mean virus level in the dust shed by that group. This relationship was similar in each of the vaccinated groups, demonstrating that measurement of the virus in dust can be used to monitor accurately both the infection status of the chickens and environmental contamination by MDV.

We have created a completely helper cell-dependent morbillivirus by modifying the genome to remove the coding sequence of the phosphoprotein (P) and recovering the recombinant virus in a cell line constitutively expressing the P protein. The P protein-deleted virus (P?) grew very inefficiently unless both of the viral accessory proteins (V and C) were also expressed. Growth of the virus was restricted to the P-expressing cell line. The P? virus grew more slowly than the parental virus and expressed much less viral protein in infected cells. The technique could be used to create virus-like particles for use as a vaccine or as antigen in immunological or serological assays.

One of the big surprises about the devastating outbreak of bluetongue serotype-8 that spread across Northern and Western Europe between 2006 and 2008 was how relatively quickly the virus was controlled and eradicated from affected countries. This was at least in part attributed to the high levels of vaccine coverage achieved in affected countries. A previous study revealed that neutralising antibodies persisted in the majority of vaccinated cattle for at least 3 years post-vaccination, indicating that cattle are likely to be protected for this time period. The current study revealed that neutralising antibodies persisted in the same group of cattle for up to 4 years post-vaccination, and that neutralising antibodies persisted for up to 2.5 years in sheep that had been vaccinated on two occasions one year apart. These results have implications for future bluetongue surveillance programmes and vaccine control strategies.

The species Corriparta virus (CORV), within the genus Orbivirus, family Reoviridae, currently contains six virus strains: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus and Jacareacanga virus. However, lack of neutralization assays, or reference genome sequence data has prevented further analysis of their intra-serogroup/species relationships and identification of individual serotypes. We report whole-genome sequence data for CORV-MRM1, which was isolated in 1960 in Australia. Comparisons of the conserved, polymerase (VP1), sub-core-shell 'T2' and core-surface 'T13' proteins encoded by genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) respectively, show that this virus groups with the other mosquito borne orbiviruses. However, highest levels of nt/aa sequence identity (75.9%/91.6% in Seg-2/T2: 77.6%/91.7% in Seg-8/T13, respectively) were detected between CORV-MRM1 and California mosquito pool virus (CMPV), an orbivirus isolated in the USA in 1974, showing that they belong to the same virus species. The data presented here identify CMPV as a member of the Corriparta virus species and will facilitate identification of additional CORV isolates, diagnostic assay design and epidemiological studies.