The use of molecular methods in studies of avian haemosporidians

During the last two decades, molecular methods to study mitochondrial DNA sequence variation have become an important part in the studies of avian haemosporidians. Up until recently, these methods have primarily been used for identification of the parasites and for tentative phylogenetic reconstructions, allowing researchers not trained in traditional parasitology to compare data across the globe. However, with the introduction of genome and transcriptome sequencing, studies are emerging that go deeper into the genetics and molecular biology of the parasites. In this chapter, we describe and summarize the common methods used for genetic barcoding of the parasites and give an introduction of what to take into account when designing a molecular study of avian haemosporidians. This chapter further discusses why nuclear genetic data are needed in order to answer several important ecological and evolutionary questions and which methods to use in order to overcome the obstacles of obtaining nuclear data of the parasites. Finally, this chapter highlights the challenges and opportunities that come with the use of molecular methods, such as how to study and interpret prevalence, the challenge of aborted developments, and how to obtain data for more robust phylogenies and population structure studies of the parasites

Transcriptome sequencing and analysis of Plasmodium gallinaceum reveals polymorphisms and selection on the apical membrane antigen-1

BACKGROUND: Plasmodium erythrocyte invasion genes play a key role in malaria parasite transmission, host-specificity and immuno-evasion. However, the evolution of the genes responsible remains understudied. Investigating these genes in avian malaria parasites, where diversity is particularly high, offers new insights into the processes that confer malaria pathogenesis. These parasites can pose a significant threat to birds and since birds play crucial ecological roles they serve as important models for disease dynamics. Comprehensive knowledge of the genetic factors involved in avian malaria parasite invasion is lacking and has been hampered by difficulties in obtaining nuclear data from avian malaria parasites. Thus the first Illumina-based de novo transcriptome sequencing and analysis of the chicken parasite Plasmodium gallinaceum was performed to assess the evolution of essential Plasmodium genes. METHODS: White leghorn chickens were inoculated intravenously with erythrocytes containing P. gallinaceum. cDNA libraries were prepared from RNA extracts collected from infected chick blood and sequencing was run on the HiSeq2000 platform. Orthologues identified by transcriptome sequencing were characterized using phylogenetic, ab initio protein modelling and comparative and population-based methods. RESULTS: Analysis of the transcriptome identified several orthologues required for intra-erythrocytic survival and erythrocyte invasion, including the rhoptry neck protein 2 (RON2) and the apical membrane antigen-1 (AMA-1). Ama-1 of avian malaria parasites exhibits high levels of genetic diversity and evolves under positive diversifying selection, ostensibly due to protective host immune responses. CONCLUSION: Erythrocyte invasion by Plasmodium parasites require AMA-1 and RON2 interactions. AMA-1 and RON2 of P. gallinaceum are evolutionarily and structurally conserved, suggesting that these proteins may play essential roles for avian malaria parasites to invade host erythrocytes. In addition, host-driven selection presumably results in the high levels of genetic variation found in ama-1 of avian Plasmodium species. These findings have implications for investigating avian malaria epidemiology and population dynamics. Moreover, this work highlights the P. gallinaceum transcriptome as an important public resource for investigating the diversity and evolution of essential Plasmodium genes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1475-2875-13-382) contains supplementary material, which is available to authorized users

Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex

Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine