Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines

Experiment 3

Data and R code for Fig3_and_S

Data from: Imperfect vaccination can enhance the transmission of highly virulent pathogens

Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek's disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by direct vaccination or by maternal vaccination prolongs host survival but does not prevent infection, viral replication or transmission, thus extending the infectious periods of strains otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent transmission can create conditions that promote the emergence of pathogen strains that cause more severe disease in unvaccinated hosts

Experiment 4

Data and R code for Fig

Experiment 2

Data and R code for Fig2_and_S

Maternal vaccination enhances viral shedding and onward transmission of hyperpathogenic MDV.

<p>Experiment 3. Groups of ten unvaccinated chicks produced by hens that were Rispens-vaccinated (solid lines) or not (dotted lines) were infected with viral strains HPRS-B14 (black) or 675A (red) and cohoused with sentinels of the same maternal antibody status. Maternally derived antibodies prolonged the survival of experimentally infected birds (<b>A</b>) and enhanced the amount of virus shed into the environment by the hyperpathogenic strain (<b>B</b>), making possible the infection of sentinels with the most virulent strain (<b>C</b>), which led to their death (<b>D</b>). Shaded regions represent 95% c.i. for unvaccinated (light) and vaccinated (dark). Raw data can be found at <a href="http://dx.doi.org/10.5061/dryad.4tn48" target="_blank">http://dx.doi.org/10.5061/dryad.4tn48</a>.</p

Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens

<div><p>Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek's disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by direct vaccination or by maternal vaccination prolongs host survival but does not prevent infection, viral replication or transmission, thus extending the infectious periods of strains otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent transmission can create conditions that promote the emergence of pathogen strains that cause more severe disease in unvaccinated hosts.</p></div

Impact of vaccination on mortality and viral shedding of five strains of MDV.

<p>Experiment 1. Groups of 20 Rhode Island Red chickens were unvaccinated (dotted lines, light shading) or HVT-vaccinated (solid lines, dark shading) at 1 d of age and challenged with viral strains HPRS-B14 (black), 571 (purple), 595 (green), Md5 (blue), or 675A (red) 8 d later. Viral strains vary in virulence in unvaccinated hosts, and vaccination protects against death (<b>top panels</b>, with strains arranged in order of increasing virulence from left to right.). Vaccination suppresses the concentration of virus in dust, but by keeping hosts alive, prolongs the infectious periods of hyperpathogenic MDV (<b>middle panels</b>). This means that cumulative number of virus genome copies (VCN) shed per bird is suppressed by vaccination for the least virulent strain and enhanced by several orders of magnitude for the most virulent (<b>bottom panels</b>). Error bars and shaded regions indicate 95% confidence interval (c.i.) Raw data can be found at <a href="http://dx.doi.org/10.5061/dryad.4tn48" target="_blank">http://dx.doi.org/10.5061/dryad.4tn48</a>.</p

Vaccination enhances transmission of hyperpathogenic MDV.

<p>Experiment 2. Groups of ten birds were HVT-vaccinated (solid lines) or not (dotted lines) and experimentally infected with one of our three most virulent MDV strains, 595 (green), Md5 (blue) and 675A (red), and co-housed with ten unvaccinated sentinel birds. Vaccination prolonged the survival of experimentally infected birds (<b>A</b>), ensuring that sentinel birds became infected (<b>B</b>) and, hence, died (<b>C</b>). In B and C, solid lines denote sentinels cohoused with vaccinated experimentally infected birds and dotted lines denote sentinels cohoused with unvaccinated experimentally infected birds. Raw data can be found at <a href="http://dx.doi.org/10.5061/dryad.4tn48" target="_blank">http://dx.doi.org/10.5061/dryad.4tn48</a>.</p