The ER-membrane transport system is critical for intercellular trafficking of the NSm movement protein and Tomato Spotted Wilt Tospovirus

Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV

Evaluation of the routine implementation of pulse oximeters into integrated management of childhood illness (IMCI) guidelines at primary health care level in West Africa : the AIRE mixed-methods research protocol

Background: The AIRE operational project will evaluate the implementation of the routine Pulse Oximeter (PO) use in the integrated management of childhood illness (IMCI) strategy for children under-5 in primary health care centers (PHC) in West Africa. The introduction of PO should promote the accurate identification of hypoxemia (pulse blood oxygen saturation Sp02 < 90%) among all severe IMCI cases (respiratory and non-respiratory) to prompt their effective case management (oxygen, antibiotics and other required treatments) at hospital. We seek to understand how the routine use of PO integrated in IMCI outpatients works (or not), for whom, in what contexts and with what outcomes.Methods: The AIRE project is being implemented from 03/2020 to 12/2022 in 202 PHCs in four West African countries (Burkina Faso, Guinea, Mali, Niger) including 16 research PHCs (four per country). The research protocol will assess three complementary components using mixed quantitative and qualitative methods: a) context based on repeated cross-sectional surveys: baseline and aggregated monthly data from all PHCs on infrastructure, staffing, accessibility, equipment, PO use, severe cases and care; b) the process across PHCs by assessing acceptability, fidelity, implementation challenges and realistic evaluation, and c) individual outcomes in the research PHCs: all children under-5 attending IMCI clinics, eligible for PO use will be included with parental consent in a cross-sectional study. Among them, severe IMCI cases will be followed in a prospective cohort to assess their health status at 14 days. We will analyze pathways, patterns of care, and costs of care.Discussion: This research will identify challenges to the systematic implementation of PO in IMCI consultations, such as health workers practices, frequent turnover, quality of care, etc. Further research will be needed to fully address key questions such as the best time to introduce PO into the IMCI process, the best SpO2 threshold for deciding on hospital referral, and assessing the cost-effectiveness of PO use. The AIRE research will provide health policy makers in West Africa with sufficient evidence on the context, process and outcomes of using PO integrated into IMCI to promote scale-up in all PHCs.Trial registration: Trial registration number: PACTR202206525204526 retrospectively registered on 06/15/202

Plasmodesmata: Channels for Viruses on the Move

The symplastic communication network established by plasmodesmata (PD) and connected phloem provides an essential pathway for spatiotemporal intercellular signaling in plant development but is also exploited by viruses for moving their genomes between cells in order to infect plants systemically. Virus movement depends on virus-encoded movement proteins (MPs) that target PD and therefore represent important keys to the cellular mechanisms underlying the intercellular trafficking of viruses and other macromolecules. Viruses and their MPs have evolved different mechanisms for intracellular transport and interaction with PD. Some viruses move from cell to cell by interacting with cellular mechanisms that control the size exclusion limit of PD whereas other viruses alter the PD architecture through assembly of specialized transport structures within the channel. Some viruses move between cells in the form of assembled virus particles whereas other viruses may interact with nucleic acid transport mechanisms to move their genomes in a non-encapsidated form. Moreover, whereas several viruses rely on the secretory pathway to target PD, other viruses interact with the cortical endoplasmic reticulum and associated cytoskeleton to spread infection. This chapter provides an introduction into viruses and their role in studying the diverse cellular mechanisms involved in intercellular PD-mediated macromolecular trafficking