Effects of Rispens CVI988 vaccination followed by challenge with Marek's disease viruses of differing virulence on the replication kinetics and shedding of the vaccine and challenge viruses.

Vaccination with "imperfect" vaccines that prevent disease but not infection is strongly implicated in the observed increased virulence of Marek's disease virus (MDV) over the past six decades. The current "gold standard" vaccine, Rispens CVI988 (Rispens), has maintained efficacy despite use for five decades, raising the question of whether it too favours higher virulence MDVs. To investigate this, we studied the kinetics of Rispens CVI988 (Rispens) and two MDV strains of different virulence in 236 commercial ISA Brown chickens vaccinated with Rispens at hatch and challenged with vMDV isolate MPF57 or vvMDV isolate FT158 on day five. Each treatment was replicated in two isolators and from 7 to 56 days post infection (dpi) peripheral blood leucocytes (PBL), feather and dust samples were collected and subjected to differential quantitative PCR (qPCR). Rispens vaccination significantly reduced challenge MDV viral load in a sample-dependant manner with evidence of a differentially greater inhibitory effect on the less virulent MDV. Similarly, challenge with the more virulent MDV reduced the Rispens viral load in PBL. Rispens virus load displayed a distinctive pattern of viral load that was similar in PBL and feathers, but different in dust. The clear effects of vaccination and challenge evident in PBL and feather samples were less clearly reflected in dust samples. The data are consistent with the Rispens vaccine reducing replication of lesser virulent MDVs to a greater extent like the HVT vaccine. Likely reasons for the persistent efficacy of Rispens vaccine are discussed.

Protection provided by Rispens CVI988 vaccine against Marek's disease virus isolates of different pathotypes and early prediction of vaccine take and MD outcome.

We tested the level of protection provided by the Rispens CVI988 (Rispens) vaccine against challenge with a virulent Marek's disease virus (MDV) pathotype (vMDV) and a very virulent pathotype (vvMDV) and the accuracy of a range of predictive measures of Marek's disease (MD) incidence and vaccine take. Commercial layer chicks (n = 236) were vaccinated (or not) with 4000 plaque-forming units (pfu) of Rispens vaccine at hatch and challenged (or not) with 500 pfu of each challenge virus five days post vaccination. The vvMDV pathotype FT158 induced higher MD incidence (65%) and mortality (33%) when compared with the vMDV pathotype MPF57 (39% and 8%, respectively). The protective index provided by the Rispens vaccine against FT158 (61%) did not differ significantly from that against MPF57 (66%). This provides additional evidence that protection provided by the Rispens vaccine is not influenced by pathotype determined in studies using vaccines of other Mardivirus species. The challenge viruses did not differ in MDV or Rispens viral load in spleen at 14 dpc (days post challenge) determined by specific quantitative polymerase chain reaction test. MDV load in peripheral blood leucocytes at 7 and 14 dpc, splenocytes at 14 dpc, feather cells at 14 and 21 dpc and isolator dust at 21 dpc were significant early indicators of subsequent MD incidence to 56 dpc. These are potentially useful as the sampling can be carried out well before the onset of MD and some measures are non-invasive. The Rispens viral load in both invasive and non-invasive samples was more useful as a measure of vaccine take.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1.

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

Replication kinetics and shedding of very virulent Marek's disease virus and vaccinal Rispens/CVI988 virus during single and mixed infections varying in order and interval between infections.

Vaccination is thought to contribute to an evolution in virulence of the Marek's disease virus (MDV) as vaccines prevent disease but not infection. We investigated the effects of co-infections at various intervals between Rispens/CVI988 vaccine virus (Rispens) and very virulent MDV (vvMDV) on the replication and shedding of each virus. The experiment used 600 ISA Brown layer chickens in 24 isolators with all treatments replicated in two isolators. Chickens were vaccinated with Rispens and/or challenged with the vvMDV isolate 02LAR on days 0, 5, or 10 post hatching providing vaccination to challenge intervals (VCI) of -10, -5, 0, 5 or 10 days with the negative values indicating challenge prior to vaccination. Peripheral blood lymphocytes (PBL), feathers and isolator exhaust dust were sampled between 7 and 56 days post infection (dpi) and subjected to quantitative real-time polymerase chain reaction (qPCR) to differentiate the two viruses. Overall Rispens significantly reduced the viral load of vvMDV in PBL and feather cells and shedding in dust. Similarly vvMDV significantly reduced the viral load of Rispens in PBL and feather cells but not in dust. VCI significantly influenced these relationships having strong positive and negative associations with load of vvMDV and Rispens respectively. Differences between the two viruses and their effects on each other were greatest in PBL and feathers, and least in dust. This study expands our understanding of the interaction between pathogenic and vaccinal viruses following vaccination with imperfect vaccines and has implications for selection of appropriate samples to test for vaccination success.

Vaccination and reduced cohort duration can drive virulence evolution: Marek's disease virus and industrialized agriculture.

Marek's disease virus (MDV), a commercially important disease of poultry, has become substantially more virulent over the last 60 years. This evolution was presumably a consequence of changes in virus ecology associated with the intensification of the poultry industry. Here, we assess whether vaccination or reduced host life span could have generated natural selection, which favored more virulent strains. Using previously published experimental data, we estimated viral fitness under a range of cohort durations and vaccine treatments on broiler farms. We found that viral fitness maximized at intermediate virulence, as a result of a trade-off between virulence and transmission previously reported. Our results suggest that vaccination, acting on this trade-off, could have led to the evolution of increased virulence. By keeping the host alive, vaccination prolongs infectious periods of virulent strains. Improvements in host genetics and nutrition, which reduced broiler life spans below 50 days, could have also increased the virulence of the circulating MDV strains because shortened cohort duration reduces the impact of host death on viral fitness. These results illustrate the dramatic impact anthropogenic change can potentially have on pathogen virulence.

Relationship between Marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical Marek's disease in broiler chickens.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.