Apicomplexans are obligate intracellular parasites that scavenge essential nutrients from their hosts via transporter proteins on their plasma membrane. The identities of the transporters that mediate amino acid uptake into apicomplexans are unknown. Here we demonstrate that members of an apicomplexan-specific protein family—the Novel Putative Transporters (NPTs)—play key roles in the uptake of cationic amino acids. We show that an NPT from Toxoplasma gondii (TgNPT1) is a selective arginine transporter that is essential for parasite survival and virulence. We also demonstrate that a homologue of TgNPT1 from the malaria parasite Plasmodium berghei (PbNPT1), shown previously to be essential for the sexual gametocyte stage of the parasite, is a cationic amino acid transporter. This reveals a role for cationic amino acid scavenging in gametocyte biology. Our study demonstrates a critical role for amino acid transporters in the survival, virulence and life cycle progression of these parasites.

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(14), Jamaica, and the Dominican Republic (13, 17). The rapid geographic expansion of the virus is attributed to movement by viremic birds during local and migratory flight behavior. To date, there is no effective drug treatment against WN virus infection and surveillance and mosquito control measures have not significantly influenced the number of human infections (27). A vaccine against WN virus represents an important approach to the prevention and control of this emerging disease.The ChimeriVax technology has been successfully used to develop a live vaccine against Japanese encephalitis (JE) virus that is now in phase II trials (23). JE virus is a close genetic relative of WN virus (31), a fact that expedited use of this technology to develop multiple WN virus vaccine candidates. The ChimeriVax technology employs the yellow fever (YF) 17D vaccine capsid and nonstructural genes to deliver the envelope genes (prM and E) of other flaviviruses. In the work presented here, the envelope genes of YF 17D were replaced with the corresponding genes of the wild-type WN virus NY99 strain previously described by Lanciotti et al. (19). The resulting YF/WN chimera lacked the mouse neuroinvasive property of WN virus and is less neurovirulent than YF 17D vaccine in both mouse and monkey models. Because WN virus, like other flaviviruses in the genus, is neurotropic for mammals (21, 29), attenuating point mutations were later introduced in the envelope of the YF/WN chimera to further reduce its virulence. Mutation sites were targeted only to regions of the envelope (E) protein gene and were based on previous observations by others (1,3,28,32) pertaining to attenuation phenotypes in related flaviviruses: specifically JE and tick-borne encephalitis viruses. Site-directed mutations in the WN virus E gene of the chimeric prototype vaccine, ChimeriVax-West Nile 01 , (ChimeriVax-WN 01 ) resulted in a significant reduction in virus neurovirulence. Here we discuss a vaccine in a YF vaccine backbone; the WN virus envelope (E) protein mutagenesis rationale; and the assessment of the safety, immunogenicity, efficacy, and genetic stability of these ChimeriVax-WN vaccine candidates in the mouse and macaque models. MATERIALS AND METHODSYF/WN chimeric clones and molecular procedures for virus assembly. Chimeric flaviviruses were constructed with the ChimeriVax two-plasmid technology previously described (9). Briefly, the two-plasmid system provides plasmid stability in Escherichia coli by dividing the cloned YF backbone into two plasmids. This provides smaller plasmids that are more stable to manipulate the YF sequences facilitating replacement of the prM and E genes of the flavivirus target vaccine. The WN virus prM and E genes used were cloned from the WN flamingo isolate 383-99 sequence (GenBank accession no. AF196835; kindly provided by John Roehrig, Centers for Disease Control and Prevention, Fort Collins, Colo.). Virus prME sequence cDNA was obtained by reverse transcription-PCR (RT-PCR) (XL-PCR kit; Applied Biosystems, Foster City, C...

We previously reported construction of a chimeric yellow fever-dengue type 2 virus (YF/DEN2) and determined its safety and protective efficacy in rhesus monkeys (F. Guirakhoo et al., J. Virol. 74:5477-5485, 2000). In this paper, we describe construction of three additional YF/DEN chimeras using premembrane (prM) and envelope (E) genes of wild-type (WT) clinical isolates: DEN1 (strain PUO359, isolated in 1980 in Thailand), DEN3 (strain PaH881/88, isolated in 1988 in Thailand), and DEN4 (strain 1228, isolated in 1978 in Indonesia). These chimeric viruses (YF/DEN1, YF/DEN3, and YF/DEN4) replicated to ϳ7.5 log 10 PFU/ml in Vero cells, were not neurovirulent in 3-to 4-week-old ICR mice inoculated by the intracerebral route, and were immunogenic in monkeys. All rhesus monkeys inoculated subcutaneously with one dose of these chimeric viruses (as monovalent or tetravalent formulation) developed viremia with magnitudes similar to that of the YF 17D vaccine strain (YF-VAX) but significantly lower than those of their parent WT viruses. Eight of nine monkeys inoculated with monovalent YF/DEN1 -3, or -4 vaccine and six of six monkeys inoculated with tetravalent YF/DEN1-4 vaccine seroconverted after a single dose. When monkeys were boosted with a tetravalent YF/ DEN1-4 dose 6 months later, four of nine monkeys in the monovalent YF/DEN groups developed low levels of viremia, whereas no viremia was detected in any animals previously inoculated with either YF/DEN1-4 vaccine or WT DEN virus. An anamnestic response was observed in all monkeys after the second dose. No statistically significant difference in levels of neutralizing antibodies was observed between YF virus-immune and nonimmune monkeys which received the tetravalent YF/DEN1-4 vaccine or between tetravalent YF/DEN1-4-immune and nonimmune monkeys which received the YF-VAX. However, preimmune monkeys developed either no detectable viremia or a level of viremia lower than that in nonimmune controls. This is the first recombinant tetravalent dengue vaccine successfully evaluated in nonhuman primates.

BackgroundThe protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite’s sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission.ResultsComparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered.ConclusionsThe need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1298-6) contains supplementary material, which is available to authorized users.

ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X7 receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X7 receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1β and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X7 receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X7 receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X7 receptor, underscoring its importance in infectious disease.

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.

Conjugates of gold nanoparticles and antibodies have useful functionalities. Here, we show how they can be used to selectively target and destroy parasitic protozoans. Gold nanorods were conjugated with an anti-Toxoplasma gondii antibody and used to target the extracellular tachyzoite which is an infectious form of an obligate parasite Toxoplasma gondii. Subsequent laser irradiation was used to kill the targeted protozoans. This concept provides a new paradigm for the treatment of parasitic protozoans.