Plasmodium parasites mount an arrest response to dihydroartemisinin, as revealed by whole transcriptome shotgun sequencing (RNA-seq) and microarray study

RNA-seq data analysis from DHA treatment of P. falciparum Limma results from 1 h treatments with 500 nM DHA in P. falciparum K1 rings, trophozoites and schizonts. (XLS 2040 kb

The Plasmodium berghei RC strain is highly diverged and harbors putatively novel drug resistance variants

Background The current first line drugs for treating uncomplicated malaria are artemisinin (ART) combination therapies. However, Plasmodium falciparum parasites resistant to ART and partner drugs are spreading, which threatens malaria control efforts. Rodent malaria species are useful models for understanding antimalarial resistance, in particular genetic variants responsible for cross resistance to different compounds. Methods The Plasmodium berghei RC strain (PbRC) is described as resistant to different antimalarials, including chloroquine (CQ) and ART. In an attempt to identify the genetic basis for the antimalarial resistance trait in PbRC, its genome was sequenced and compared with five other previously sequenced P. berghei strains. Results We found that PbRC is eight-fold less sensitive to the ART derivative artesunate than the reference strain PbANKA. The genome of PbRC is markedly different from other strains, and 6,974 single nucleotide variants private to PbRC were identified. Among these PbRC private variants, non-synonymous changes were identified in genes known to modulate antimalarial sensitivity in rodent malaria species, including notably the ubiquitin carboxyl-terminal hydrolase 1 gene. However, no variants were found in some genes with strong evidence of association with ART resistance in P. falciparum such as K13 propeller protein. Discussion The variants identified in PbRC provide insight into P. berghei genome diversity and genetic factors that could modulate CQ and ART resistance in Plasmodium spp

In Vitro and In Vivo Attenuation of Vesicular Stomatitis Virus (VSV) by Phosphoprotein Deletion.

Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication

Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing <i>Pasteurella multocida</i> OmpH Protein for Fowl Cholera Prevention in Ducks

Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases

Induction of immune responses following immunization with VSV-ΔP expressing H1 and H7 HA.

<p>BALB/c mice (5 mice/group) were intravenously injected with 1×10⁷ pfu of VSVs in 100 μl at days 0 and 21. At day 28, sera were harvested to determine for H1N1-specific and H7N9-specific IgG levels at a titer of 5,120. Values are averages of two independent experiments with error bars showing SD.</p

Decreased lethality in mice after intracranial injection with VSV-ΔP.

<p>ICR mice (5 mice/group) were lightly anesthetized with ether and then intracranially injected with either PBS or 1×10⁴ pfu of VSVs. (A) Body weight was measured daily and (B) survival was plotted using the Kaplan-Meier survival curve. Values are averages of five mice with error bars showing SD and are representative of two independent experiments. NS, not significant; *, p<0.5; **, p<0.05.</p

P gene deletion attenuated replication of recombinant virus.

<p>(A) BHK-21 and BHK-P cells were infected with VSV-ΔP at an MOI of 1 and observed for cytopathic effects (CPE). Infected cells were then subjected to flow cytometry to quantify the percentage of mCherry-expressing cells. The pictures are representative of triplicate samples. (B) BHK-P cells were infected with VSV or VSV-ΔP at an MOI of 0.01. Supernatants were harvested at the indicated time points for plaque assays. Values are averages of two independent experiments with error bars showing standard deviation (SD). (C) Viruses were serially diluted for plaque titration, and plaques were stained with neutral red for visualization. Representative images of VSV and VSV-ΔP were selected for plaque size comparison.</p

Construction of recombinant VSVs with P gene deletion.

<p>(A) The top schematic shows the VSV genome layout and the naturally occurring restriction sites used for cloning. To construct VSV-ΔP, the VSV genome was digested with <i>Eco</i>RV, religated in the absence of the P gene fragment and the mCherry gene was then inserted between the G and L genes. The mCherry gene was replaced by HN, H1 or H7 HA to generate VSV-ΔP-HN, VSV-ΔP-HA1 and VSV-ΔP-HA7, respectively. (B) Supporting cells (BHK-P) were constructed by transducing BHK-21 cells with lentivirus bearing the P gene, and the selected clone expressing the emerald fluorescent protein was examined by (C) bright field and (D) fluorescence imaging. (E) BHK-P cells were infected with VSV or VSV-ΔP, and supernatants were harvested for viral genome extraction and RT-PCR. RBZ, hepatitis virus delta ribozyme; T7, T7 RNA polymerase leader; T7 ter, T7 terminator; LTR, long terminal repeat; ψ, packaging signal; RRE, rev responsive element; cPPT, central polypurine tract; SFFV, spleen focus-forming virus (promoter); WPRE, woodchuck hepatitis virus post-transcription regulatory element; ΔU3, U3 deletion.</p

Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.

<p>Virus neutralizing (VN) assay and hemagglutination inhibition (HAI) assay.</p