Application of high-throughput sequencing for the analyses of PRRSV-host interactions

Doctor of PhilosophyDepartment of Diagnostic Medicine and PathobiologyRaymond R. R. RowlandPorcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most costly virus to the swine industry, worldwide. This study explored the application of deep sequencing techniques to understand better the virus-host interaction. On the virus side, PRRSV exists as a quasispecies. The first application of deep sequencing was to investigate amino acid substitutions in hypervariable regions during acute infection and after virus rebound. The appearance and disappearance of mutations, especially the generation of a new N-glycosylation site in GP5, indicated they are likely the result of immune selection. The second application of deep sequencing was to investigate the quasispecies makeup in pigs with severe combined immunodeficiency (SCID) that lack B and T cells. The results showed the same pattern of amino acid substitutions in SCID and normal littermates and no different mutations were identified between SCID and normal littermates. This suggests the mutations that appear during the early stages of infection are the product of the virus becoming adapted to replication in pigs. The third application of deep sequencing was to investigate the locations of recombination events between GFP-expressing PRRSV infectious clones. The results identified different cross-over occurred within three conserved regions between EGFP and GFPm genes. And finally, the fourth goal was applied to develop a set of sequencing tools for analyzing the host antibody repertoire. A simple method was developed to amplify swine VDJ repertoires. Shared and abundant VDJ sequences that are likely expressed by PRRSV-activated B cells were determined in pigs that had different neutralization activities. These sequences are potentially correlated with different antibody responses

Porcine RIG-I and MDA5 Signaling CARD Domains Exert Similar Antiviral Function Against Different Viruses

The RIG-I-like receptors (RLRs) RIG-I and MDA5 play critical roles in sensing and fighting viral infections. Although RIG-I and MDA5 have similar molecular structures, these two receptors have distinct features during activation. Further, the signaling domains of the N terminal CARD domains (CARDs) in RIG-I and MDA5 share poor similarity. Therefore, we wonder whether the CARDs of RIG-I and MDA5 play similar roles in signaling and antiviral function. Here we expressed porcine RIG-I and MDA5 CARDs in 293T cells and porcine alveolar macrophages and found that MDA5 CARDs exhibit higher expression and stronger signaling activity than RIG-I CARDs. Nevertheless, both RIG-I and MDA5 CARDs exert comparable antiviral function against several viruses. Transcriptome analysis showed that MDA5 CARDs are more effective in regulating downstream genes. However, in the presence of virus, both RIG-I and MDA5 CARDs exhibit similar effects on downstream gene transcriptions, reflecting their antiviral function

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

Background. Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods. The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results. Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions. The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation

Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034

Crystal structures of protease/inhibitor complexes guided optimization of the buried nonpolar surface area thereby maximizing hydrophobic binding. The resulting potent tripeptide inhibitor is in clinical trials

Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin induced dormant ring stages of Plasmodium falciparum

Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART induced ring stage dormancy and recovery has been implicated as possible cause of recrudescence; however, little is known about the characteristics of dormant parasites including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA) induced dormancy and recovery. Transcription analysis showed an immediate down regulation for 10 genes following exposure to DHA, but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, were also maintained. Additions of inhibitors for biotin acetyl CoA carbozylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively following DHA treatment. Our results demonstrate most metabolic pathways are down regulated in DHA induced dormant parasites. In contrast fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment

Mitochondrial membrane potential in a small subset of Artemisinin-induced dormant Plasmodium falciparum parasites in vitro

Artemisinin induced dormancy is a proposed mechanism for failures of mono-therapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that following dihydroartemisinin (DHA) treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential (MMP) marker, and persisted to recovery. FACS sorted RH-positive parasites resumed growth at 10,000/well while RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was only detected in RH-positive dormant parasites. Importantly, after treating dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage

Relapses of Plasmodium vivax infection result from clonal hypnozoites activated at predetermined intervals

Plasmodium vivax infections are characterized by varying numbers of relapses occurring at different intervals as a result of activation of liver-stage hypnozoites. Parasite or host factors that determine the number and timing of relapses are unclear. In the present article, we report the analysis of relapse patterns and molecular characterization of parasites collected from Australian soldiers experiencing relapses of vivax malaria after exposure in East Timor. Although high molecular diversity was observed, a single allelic type was identified in association with 99% of relapses. Importantly, in 71% of patients experiencing >1 relapse, the allelic types were clonal and different in the 2 different relapses. These results, combined with those from a computer simulation model, suggest that a single hypnozoite clone was activated, causing a relapse, and that multiple relapses most likely arose from coordinated activation of hypnozoites originating from different parasite strains. These findings suggest remarkable regulation of relapse intervals in vivax malaria.</p

Cytochrome P450 2D6 profiles and anti-relapse efficacy of tafenoquine against Plasmodium vivax in Australian Defence Force personnel

Plasmodium vivax infections and relapses remain a major health problem for malaria-endemic countries, deployed military personnel, and travelers. Presumptive anti-relapse therapy and radical cure using the 8-aminoquinoline drugs primaquine and tafenoquine are necessary to prevent relapses. Although it has been demonstrated that the efficacy of primaquine is associated with Cytochrome P450 2D6 (CYP2D6) activity, there is insufficient data on the role of CYP2D6 in the anti-relapse efficacy of tafenoquine. We investigated the relationship between CYP2D6 activity status and tafenoquine efficacy in preventing P. vivax relapses retrospectively using plasma samples collected from Australian Defence Force personnel deployed to Papua New Guinea and Timor-Leste who participated in clinical trials of tafenoquine during 1999–2001. The CYP2D6 gene was amplified from plasma samples and fully sequenced from 92 participant samples, comprised of relapse (n = 31) and non-relapse (n = 61) samples, revealing 14 different alleles. CYP2D6 phenotypes deduced from combinations of CYP2D6 alleles predicted that among 92 participants 67, 15, and 10 were normal, intermediate, and poor metabolizers, respectively. The deduced CYP2D6 phenotype did not correlate with the corresponding participant’s plasma tafenoquine concentrations that were determined in the early 2000s by high-performance liquid chromatography or liquid chromatography-mass spectrometry. Furthermore, the deduced CYP2D6 phenotype did not associate with P. vivax relapse outcomes. Our results indicate that CYP2D6 does not affect plasma tafenoquine concentrations and the efficacy of tafenoquine in preventing P. vivax relapses in the assessed Australian Defence Force personnel