抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

BackgroundCoronavirus disease 2019 (Covid-19) is associated with diffuse lung damage. Glucocorticoids may modulate inflammation-mediated lung injury and thereby reduce progression to respiratory failure and death.MethodsIn this controlled, open-label trial comparing a range of possible treatments in patients who were hospitalized with Covid-19, we randomly assigned patients to receive oral or intravenous dexamethasone (at a dose of 6 mg once daily) for up to 10 days or to receive usual care alone. The primary outcome was 28-day mortality. Here, we report the final results of this assessment.ResultsA total of 2104 patients were assigned to receive dexamethasone and 4321 to receive usual care. Overall, 482 patients (22.9%) in the dexamethasone group and 1110 patients (25.7%) in the usual care group died within 28 days after randomization (age-adjusted rate ratio, 0.83; 95% confidence interval [CI], 0.75 to 0.93; P<0.001). The proportional and absolute between-group differences in mortality varied considerably according to the level of respiratory support that the patients were receiving at the time of randomization. In the dexamethasone group, the incidence of death was lower than that in the usual care group among patients receiving invasive mechanical ventilation (29.3% vs. 41.4%; rate ratio, 0.64; 95% CI, 0.51 to 0.81) and among those receiving oxygen without invasive mechanical ventilation (23.3% vs. 26.2%; rate ratio, 0.82; 95% CI, 0.72 to 0.94) but not among those who were receiving no respiratory support at randomization (17.8% vs. 14.0%; rate ratio, 1.19; 95% CI, 0.92 to 1.55).ConclusionsIn patients hospitalized with Covid-19, the use of dexamethasone resulted in lower 28-day mortality among those who were receiving either invasive mechanical ventilation or oxygen alone at randomization but not among those receiving no respiratory support. (Funded by the Medical Research Council and National Institute for Health Research and others; RECOVERY ClinicalTrials.gov number, NCT04381936. opens in new tab; ISRCTN number, 50189673. opens in new tab.)

/pdf/dexamethasone-in-hospitalized-patients-with-covid-19-2p8rrjo50m.pdf

Dexamethasone in Hospitalized Patients with Covid-19

Evolution and ecology of influenza A viruses.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses

A recent theory suggests that the evaporation kinetics of macromolecular solutions may be insensitive to the ambient relative humidity (RH) due to the formation of a ‘polarisation layer’ of solutes at the air-solution interface. We study the evaporation of polyvinyl alcohol solutions from an open-ended capillary, and ﬁnd RH-insensitive evaporation up to RH ≈ 85%. We also conﬁrm the predicted decrease in evaporation rate over time. However, RH-insensitive evaporation sets in earlier than predicted by theory, suggesting additional polymer-interface interactions. We discuss the relevance of these observations for respiratory virus transmission via aerosols.

The evaporation of concentrated polymer solutions is insensitive to relative humidity

: Evidence suggests that susceptibility to avian inﬂuenza A virus in chickens is inﬂuenced by host genetics, but the mechanisms are poorly understood. A previous study demonstrated that inbred line 0 chickens are more resistant to low-pathogenicity avian inﬂuenza (LPAI) infection than line CB.12 birds based on viral shedding, but the resistance was not associated with higher AIV-speciﬁc IFN γ responses or antibody titres. In this study, we investigated the proportions and cytotoxic capacity of T-cell subpopulations in the spleen and the early immune responses in the respiratory tract, analysing the innate immune transcriptome of lung-derived macrophages following in vitro stimulation with LPAI H7N1 or the TLR7 agonist R848. The more susceptible C.B12 line had a higher proportion of CD8 αβ + γδ and CD4 + CD8 αα + α V β 1 T cells, and a signiﬁcantly higher proportion of the CD8 αβ + γδ and CD8 αβ + α V β 1 T cells expressed CD107a, a surrogate marker of degranulation. Lung macrophages isolated from line C.B12 birds expressed higher levels of the negative regulator genes TRIM29 and IL17REL, whereas macrophages from line 0 birds expressed higher levels of antiviral genes including IRF10 and IRG1 . After stimulation with R848, the macrophages from line 0 birds mounted a higher response compared to line C.B12 cells. Together, the higher proportion of unconventional T cells, the higher level of cytotoxic cell degranulation ex vivo and post-stimulation and the lower levels of antiviral gene expression suggest a potential role of immunopathology in mediating susceptibility in C.B12 birds.

Explorer Comparative analysis of different inbred chicken lines highlights how a hereditary inflammatory state affects susceptibility to avian influenza virus

The disclosure relates to mutant recombinant influenza virus gene segment 7 with at least one mutation that modulates expression of M2 and M42 polypeptide. Also disclosed are recombinant influenza viruses comprising the mutant influenza virus gene segment 7, compositions comprising the mutant recombinant influenza virus gene segment 7, use of such mutant recombinant influenza virus gene segment 7 and mutant recombinant Influenza viruses.

Influenza Virus Mutants and Uses Therefor

Human rotavirus (RV) vaccines used worldwide have been developed using live attenuated platforms. The recent development of a reverse genetics system for RVs has delivered the possibility of engineering chimeric viruses expressing heterologous peptides from other virus species to generate polyvalent vaccines. We tested the feasibility of this using two approaches. Firstly, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian SA11 RV strain viral protein (VP) 4. Secondly, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C-terminus of non-structural protein (NSP) 3 of the bovine RF strain RV, with or without an intervening T2A peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RF strain. Except for the RBD mutant, all NSP3 mutants delivered endpoint titres and replication kinetics comparable to that of the WT virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants but only RBM mutant showed cross reactivity with SARS-CoV-2 RBD antibody. The tolerability of large peptide insertions in the NSP3 segment highlights the potential for this approach in the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an exemplar. Small SARS-CoV-2 peptide insertion (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titre and replication, thus limiting its use as a potential vaccine expression platform. To test RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids. With a T2A-separated 193 amino acid tag on NSP3, there was little effect on the viral rescue efficiency, titre and replication. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. This is the first report describing epitope tagging of VP4, and of a reverse genetics system for the RF strain. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

Paul Digard

Papers

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses

The evaporation of concentrated polymer solutions is insensitive to relative humidity

Explorer Comparative analysis of different inbred chicken lines highlights how a hereditary inflammatory state affects susceptibility to avian influenza virus

Influenza Virus Mutants and Uses Therefor

Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes