Differential quantification of cloned CVI988 vaccine strain and virulent RB-1B strain of Marek's disease viruses in chicken tissues, using real-time PCR.

The 'gold standard' vaccine against Marek's disease in poultry is the CVI988/Rispens virus, which is not easily distinguishable, antigenically or genetically, from virulent Marek's disease herpesvirus. Accurate differential measurement of the CVI988 vaccine and virulent viruses is important to investigate mechanisms of vaccinal protection. Minimal sequence differences between CVI988 and virulent MDV strains restrict the application of molecular diagnostic methods such as real-time PCR to distinguish between these viruses. The use of bacterial-artificial-chromosome (BAC) cloned CVI988 virus, which carries the BAC vector sequences in place of the U(s)2 gene, allows its differential quantification from virulent strains using real-time PCR assays that target the BAC vector sequence and the U(S)2 gene respectively. These novel assays allowed investigation of replication of both serotype-1 vaccine virus (cloned CVI988) and challenge virus (RB-1B strain) in tissues of individual chickens in an experimental vaccination-challenge model of Marek's disease.

Comparative efficacy of BAC-derived recombinant SB-1 vaccine and the parent wild type strain in preventing replication, shedding and disease induced by virulent Marek's disease virus.

A widely used vaccine against Marek's disease (MD) in poultry is the virus SB-1, which is antigenically-related to the causative agent, Marek's disease herpesvirus. We recently cloned the SB-1 genome as an infectious bacterial artificial chromosome, BAC, (pSB-1). The protective efficacies and replication kinetics of pSB-1 and the parent strain (SB-1) were compared in an experimental model of MD induced by a virulent strain, RB-1B. Although vaccine virus replication and shedding was lower for pSB-1 than for SB-1, both vaccines reduced replication and shedding of RB-1B, and were equally effective in protecting chickens against MD. With the cloning of pSB-1, we have now generated full length genomic clones of MD vaccine virus strains belonging to each of the three serotypes. Vaccine viruses derived from each of these clones demonstrated protective efficacies at levels similar to those produced by the respective parent viruses, demonstrating their suitability to be used as vaccine candidates.

Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture.

The influence on virus replication in culture of the presence and location of glycosylation sites on the haemagglutinin (HA) glycoprotein of avian influenza viruses and differences in length of the stalk region of their neuraminidase (NA) glycoprotein was examined using reassortant viruses. Plaque size was measured in the presence or absence of bacterial neuraminidase (CPNA) and/or an influenza virus NA inhibitor, zanamivir, to assess the relative contribution of the NA to replication efficiency in tissue culture. The following conclusions were drawn, (1) HA lacking glycosylation at 158 gives inefficient growth when combined with short-stalked NAs, and efficient growth when combined with long-stalked NAs. (2) Glycosylation at 158 of HA makes the virus less dependent on NA for release from its receptors. (3) HA with glycosylation at 158 gives efficient growth when combined with short-stalked NAs but, when combined with long-stalked NAs, growth is very efficient and excess NA activity is disadvantageous. (4) HA having glycosylation at 158 combined with short-stalked NAs, or HA lacking glycosylation at 158 combined with long-stalked NAs may represent optimal combinations. The results reinforce the importance of a balance of HA and NA activity for efficient virus exit from, and entry into cells.

Genetic analysis reveals that both haemagglutinin and neuraminidase determine the sensitivity of naturally occurring avian influenza viruses to zanamivir in vitro.

The basis of differential sensitivity of replication of influenza viruses to the neuraminidase-specific inhibitor zanamivir was examined using four avian influenza viruses and reassortants produced between them. IC(50) values for inhibition of neuraminidase activity by zanamivir were similar for each of the four viruses, whereas the haemagglutinating activity of each of the viruses was relatively insensitive to zanamivir. However, the four viruses showed distinct zanamivir-sensitivity profiles in tissue culture. Analysis of the reassortant viruses showed that sensitivity was determined by the haemagglutinin gene (segment 4) and the neuraminidase gene (segment 6) and was independent of the remaining six RNA segments. Decreased sensitivity to zanamivir was associated with possession of a haemagglutinin that is released from cells with decreased dependence on neuraminidase and with possession of a neuraminidase that has a short stalk region.

Development and composition of lymphoid lesions in the spleens of Marek's disease virus-infected chickens: Association with virus spread and the pathogenesis of Marek's disease.

Changes in lymphocyte distribution in spleens of Marek's disease virus (MDV) infected White Leghorn chickens of line 72 (MD susceptible) and line 61 (MD resistant) were studied by immunocytochemistry. Lymphocytes expressing the MDV antigen pp38 (predominantly B cells) were detected from 4 to 6 days post-inoculation (d.p.i.) but not at or after 8 d.p.i., and were more numerous in line 72. In line 61, infection resulted in depletion of B lymphocytes and an increase in T lymphocytes from 3 to 6 d.p.i., but no change in distribution of these cells. From 8 d.p.i., the B-dependent tissue began to recover and the T cells decreased in number. In line 72, infection caused a dramatic change in lymphocyte distribution, with formation of 'lymphoid lesions'. Diffuse, irregular patches of B lymphocytes, around the capillaries, became surrounded by large aggregates of TCRαß1(+) CD8(+) and CD4(+) lymphocytes, bordered by a band of TCRγδ(+) lymphocytes. From 8 d.p.i., the B-dependent areas partially recovered, while TCRαß1(+) CD4(+) and CD8(+) lymphocytes, potentially transformed, became extensively scattered throughout the spleen. We conclude that in line 72, replication and spread of MDV is more efficient and T cell responses in early infection are greater, favouring the tumour stage of the disease.

Differential susceptibility to Marek's disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes.

Flow cytometric and immunocytochemical techniques were used to quantify, identify and locate Marek's disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Marek's disease (line 7(2)) and another resistant (line 6(1)), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 6(1) and 7(2), pp38+ lymphocytes were detected at 4-6 days in the spleen, thymus and bursa. pp38+ cells could not be detected from day 8 onwards. In both lines, pp38+ lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38+ cells were predominantly B lymphocytes, but CD4+ and CD8+ TCR alphabeta+ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38+ lymphocytes was greater in line 7(2) than in line 6(1). pp38+ lymphocytes were not detected in the peripheral blood at any time. The data show that the differential susceptibility of lines 6(1) and 7(2) to the development of Marek's disease lymphoma is not attributable to differences in phenotype or location of pp38+ lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38+ lymphocytes.

A flow cytometric method for identifying Marek's disease virus pp38 expression in lymphocyte subpopulations.

Identification of Marek's disease virus-infected lymphocytes is essential for a full understanding of the pathogenesis of Marek's disease. This paper describes the development of a simple, quantitative and reproducible flow cytometric technique for the identification of the phenotype of Marek's disease virus-infected lymphocytes. The method is based upon the detection of the Marek's disease virus-specific phosphoprotein pp38, in saponin-permeabilized lymphocytes, using the monoclonal antibody BDI, and the identification of the phenotype of pp38+ lymphocytes using monoclonal antibodies against lymphocyte surface markers. pp38 expression in the spleen was demonstrated in the cytolytic phase of infection. The mean proportion of pp38+ lymphocytes at 4 days post-infection was 0.43%. Of these, 95% were B lymphocytes, while only 4% were T lymphocytes (both CD4+ and CD8+). The potential of the technique for the investigation of the cytolytic phase of Marek's disease is discussed.