Viral PIC-pocketing: RSV sequestration of translational preinitiation complexes into bi-phasic biomolecular condensates.

Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Here, we report a strategy whereby the orthopneumoviruses sequester various components of the translational preinitiation complex machinery into viral inclusion bodies to facilitate translation of their own mRNAs-PIC-pocketing. Electron microscopy of respiratory syncytial virus (RSV)-infected cells revealed bi-phasic organization of IBs, specifically, spherical "droplets" nested within the larger inclusion. Using correlative light and electron microscopy, combined with fluorescence in situ hybridization, we showed that the observed bi-phasic morphology represents functional compartmentalization of the inclusion body and that these domains are synonymous with the previously reported inclusion body-associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as components of eukaryotic translation initiation factors (eIF), eIF4F and eIF3, and 40S complexes involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates that the majority of viral mRNA translation occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however, interestingly, ribopuromycylation assays showed no changes to global levels of translation. The mechanistic basis for this pathway was subsequently determined to involve the viral M2-1 protein interacting with eIF4G, likely to facilitate its transport between the cytoplasm and the separate phases of the viral inclusion body. In summary, our data show that these viral organelles function to spatially regulate early steps in viral translation within a highly selective bi-phasic biomolecular condensate. IMPORTANCE: Respiratory syncytial viruses (RSVs) of cows and humans are a significant cause of morbidity and mortality in their respective populations. These RNA viruses replicate in the infected cells by compartmentalizing the cell's cytoplasm into distinct viral microdomains called inclusion bodies (IBs). In this paper, we show that these IBs are further compartmentalized into smaller structures that have significantly different density, as observed by electron microscopy. Within smaller intra-IB structures, we observed ribosomal components and evidence for active translation. These findings highlight that RSV may additionally compartmentalize translation to favor its own replication in the cell. These data contribute to our understanding of how RNA viruses hijack the cell to favor replication of their own genomes and may provide new targets for antiviral therapeutics in vivo.

Foot-and-mouth disease virus localisation on follicular dendritic cells and sustained induction of neutralising antibodies is dependent on binding to complement receptors (CR2/CR1).

Previous studies have shown after the resolution of acute infection and viraemia, foot-and-mouth disease virus (FMDV) capsid proteins and/or genome are localised in the light zone of germinal centres of lymphoid tissue in cattle and African buffalo. The pattern of staining for FMDV proteins was consistent with the virus binding to follicular dendritic cells (FDCs). We have now demonstrated a similar pattern of FMDV protein staining in mouse spleens after acute infection and showed FMDV proteins are colocalised with FDCs. Blocking antigen binding to complement receptor type 2 and 1 (CR2/CR1) prior to infection with FMDV significantly reduced the detection of viral proteins on FDCs and FMDV genomic RNA in spleen samples. Blocking the receptors prior to infection also significantly reduced neutralising antibody titres, through significant reduction in their avidity to the FMDV capsid. Therefore, the binding of FMDV to FDCs and sustained induction of neutralising antibody responses are dependent on FMDV binding to CR2/CR1 in mice.

Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy.

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. IMPORTANCE Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

Immune-Mediated Glycocalyx Remodeling in Hospitalized COVID-19 Patients.

PURPOSE: Vascular and immune dysfunction are hallmarks of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections and coronavirus disease 2019 (COVID-19). Although our understanding of the pathogenesis of COVID-19 has rapidly evolved, much of the focus has been on the immune mechanisms underlying COVID-19. In addition to immune dysfunction, vascular injury is also associated with COVID-19 and is a major driver of clinical deterioration in SARS-CoV-2 infections. The glycocalyx (GAC), a sugar-based shell that surrounds all mammalian cells, is an important regulator of vascular and immune responses. In sepsis, vascular dysfunction contributes to acute respiratory distress syndrome (ARDS) by altering vessel integrity, promoting thrombosis, and accelerating inflammation, all of which are also present in COVID-19. Observational studies in sepsis have found an association between levels of circulating GAC degradation products with both organ dysfunction and mortality. Although vascular dysfunction is a hallmark of COVID-19, it remains unclear whether GAC disruption occurs in COVID-19 and if GAC disruption contributes to the clinical progression of COVID-19. METHODS: In this prospective cohort study, we measured the GAC components syndecan-1 (SDC1) and hyaluronan (Hyal) along with inflammatory cytokines in 12 hospitalized COVID-19 patients and 8 healthy controls (HC). RESULTS: In agreement with other studies, we found that inflammatory cytokines are elevated in hospitalized COVID-19 patients compared with HC [median (IQR), all units picograms per milliliter: IL-6 4.65 (3.32-9.16) vs 0.69 (0.55-0.89), p < 0.001; TNFα 4.49 (1.87-8.03) vs 0.04 (0.04-0.84), p < 0.001]. Additionally, we found that the GAC components SDC1 and Hyal are also elevated in COVID-19 patients [median (IQR), all units picograms per milliliter: SDC1: 247.37 (101.43-458.26) vs 84.8 (52.88-123.59), p = 0.036; Hyal: 26.41 (16.4-35.1) vs 3.01 (1.66-4.61), p < 0.001]. CONCLUSION: We propose that GAC markers offer insights into the pathobiology of COVID-19, potentially guide therapeutic approaches, and could aid in early risk stratification that is particularly beneficial in phasic diseases such as COVID-19.

Discrete Virus Factories Form in the Cytoplasm of Cells Coinfected with Two Replication-Competent Tagged Reporter Birnaviruses That Subsequently Coalesce over Time.

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common; however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus coinfection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11 or a tetracysteine (TC) tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection or stained with a biarsenical derivative of the red fluorophore resorufin (ReAsH) following PBG98-VP1-TC infection, had green or red foci in the cytoplasm, respectively, that colocalized with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 h postinfection (hpi) (P < 0.0001), while the average area increased from 1.24 to 45.01 µm2 (P < 0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57 µm/s at 16 hpi compared to 0.22 µm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During coinfection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10 to 16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence.IMPORTANCE Reassortment is common in viruses with segmented double-stranded RNA (dsRNA) genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a coinfection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 h postinfection. We hypothesize that VF coalescence is required for the reassortment of the Birnaviridae This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

Assessing Barriers to Healthy Eating in Hospitalized Older Adults With Heart Failure: Psychometric Properties of Two Questionnaires.

BACKGROUND: Dietary sodium excess and malnutrition have been associated with poor outcomes in heart failure (HF). Few previous studies have examined the barriers to following a low-sodium, nutritionally robust diet in hospitalized patients with HF. METHODS AND RESULTS: As part of a dietary intervention pilot study, 76 inpatients with HF (age 71 ±â€¯8 years, 30% female, 30% black, 36% Hispanic/Latino) completed 2 questionnaires, the Dietary Sodium Restriction Questionnaire (DSRQ) and the Brief Dietary Psychosocial Scale (BDPS), to assess challenges in following a low-sodium, nutritionally complete diet. We assessed the factor structure of the DSRQ and BDPS with confirmatory and exploratory factor analysis (CFA and EFA). CFA did not support the established 3-factor solution for the DSRQ; instead, EFA indicated that a 2-factor solution (subjective norms/attitudes and perceived behavioral control) provided the best fit for the data. EFA supported 4 separate factors for the BDPS, as in its original derivation. Cronbach's alphas supported internal consistency reliability for both scales (DSRQ: 0.85-0.94; BDPS: 0.72-0.95). CONCLUSIONS: In a mixed-ethnicity group of hospitalized older patients with HF, the DSRQ and BDPS have reasonable psychometric properties. These questionnaires may help identify barriers to healthy dietary practices and facilitate nutritional interventions in this high-risk population.

Insufficient Calorie Intake Worsens Post-Discharge Quality of Life and Increases Readmission Burden in Heart Failure.

OBJECTIVES: The purpose of this study was to evaluate the relationship between calorie intake and post-discharge outcomes in hospitalized patients with heart failure (HF). BACKGROUND: Malnutrition increases adverse outcomes in HF, and dietary sodium restriction may inadvertently worsen nutritional intake. METHODS: In a dietary intervention trial, baseline nutritional intake in HF inpatients was estimated using the Block Food Frequency Questionnaire (FFQ), and the Nutritional Risk Index (NRI) was calculated. Insufficient calorie intake was defined as <90% of metabolic needs, and a 15-point micronutrient deficiency score was created. Adjusted linear, logistic, and negative binomial regression were used to evaluate associations between insufficient calorie intake and quality of life (using the Kansas City Cardiomyopathy Questionnaire Clinical Summary [KCCQ-CS]), readmission risk, and days rehospitalized over 12 weeks. RESULTS: Among 57 participants (70 ± 8 years of age; 31% female; mean body mass index 32 ± 8 kg/m2); median sodium and calorie intake amounts were 2,987 mg/day (interquartile range [IQR]: 2,160 to 3,540 mg/day) and 1,602 kcal/day (IQR: 1,201 to 2,142 kcal/day), respectively; 11% of these patients were screened as malnourished by the NRI. All patients consuming <2,000 mg/day sodium had insufficient calorie intake; this group also more frequently had dietary micronutrient and protein deficiencies. At 12 weeks, patients with insufficient calorie intake had less improvement in the KCCQ-CS score (ß = -14.6; 95% confidence interval [CI]: -27.3 to -1.9), higher odds of readmission (odds ratio: 14.5; 95% CI: 2.2 to 94.4), and more days rehospitalized (incident rate ratio: 31.3; 95% CI: 4.3 to 229.3). CONCLUSIONS: Despite a high prevalence for obesity and rare overt malnutrition, insufficient calorie intake was associated with poorer post-discharge quality of life and increased burden of readmission in patients with HF. Inpatient dietary assessment could improve readmission risk stratification and identify patients for nutritional intervention. (Geriatric Out of Hospital Randomized Meal Trial in Heart Failure [GOURMET-HF] NCT02148679).

Home-Delivered Meals Postdischarge From Heart Failure Hospitalization.

Background In patients with heart failure (HF), malnutrition and dietary sodium excess are common and may worsen outcomes. No prior studies have provided low-sodium, nutritionally complete meals following HF hospitalization. Methods and Results The GOURMET-HF study (Geriatric Out-of-Hospital Randomized Meal Trial in Heart Failure) randomized patients discharged from HF hospitalization to 4 weeks of home-delivered sodium-restricted Dietary Approaches to Stop Hypertension meals (DASH/SRD; 1500 mg sodium/d) versus usual care. The primary outcome was the between-group change in the Kansas City Cardiomyopathy Questionnaire summary score from discharge to 4 weeks postdischarge. Additional outcomes included changes in the Kansas City Cardiomyopathy Questionnaire clinical summary score and cardiac biomarkers. All patients were followed 12 weeks for death/all-cause readmission and potential diet-related adverse events (symptomatic hypotension, hyperkalemia, acute kidney injury). Sixty-six patients were randomized 1:1 at discharge to DASH/SRD versus usual care (age, 71±8 years; 30% female; ejection fraction, 39±18%). The Kansas City Cardiomyopathy Questionnaire summary score increased similarly between groups (DASH/SRD 46±23-59±20 versus usual care 43±19-53±24; P=0.38), but the Kansas City Cardiomyopathy Questionnaire clinical summary score increase tended to be greater in DASH/SRD participants (47±22-65±19 versus 45±20-55±26; P=0.053). Potentially diet-related adverse events were uncommon; 30-day HF readmissions (11% versus 27%; P=0.06) and days rehospitalized within that timeframe (17 versus 55; P=0.055) trended lower in DASH/SRD participants. Conclusions Home-delivered DASH/SRD after HF hospitalization appeared safe in selected patients and had directionally favorable effects on HF clinical status and 30-day readmissions. Larger studies are warranted to clarify the effects of postdischarge nutritional support in patients with HF. Clinical Trial Registration URL: https://www.clinicaltrials.gov . Unique identifier: NCT02148679.

Prisoner denied access to medical marijuana.

In a case that has recently come to the attention of the editors, the Federal Court refused to order Health Canada to provide a federal prisoner with medical marijuana, even though he possessed legal authorization to possess marijuana.

Canadian court denies prisoner access to anti-anxiety medication.

In a judgment released on 22 September 2004, the Federal Court refused to order a federal correctional institution to provide a prisoner with the anti-anxiety medication clonazepam, one among a class of medications known as benzodiazepines. Benzodiazepines are available for prescription by Correctional Service Canada (CSC) physicians and psychiatrists. However, the CSC formulary provides for a discontinuation schedule for inmates who come from the provincial penitentiary system where the continued use of benzodiazepines is not indicated.

Low-sodium dietary approaches to stop hypertension diet reduces blood pressure, arterial stiffness, and oxidative stress in hypertensive heart failure with preserved ejection fraction.

Recent studies suggest that oxidative stress and vascular dysfunction contribute to heart failure with preserved ejection fraction (HFPEF). In salt-sensitive HFPEF animal models, diets low in sodium and high in potassium, calcium, magnesium, and antioxidants attenuate oxidative stress and cardiovascular damage. We hypothesized that the sodium-restricted Dietary Approaches to Stop Hypertension diet (DASH/SRD) would have similar effects in human hypertensive HFPEF. Thirteen patients with treated hypertension and compensated HFPEF consumed the DASH/SRD for 21 days (all food/most beverages provided). The DASH/SRD reduced clinic systolic (155-138 mm Hg; P=0.02) and diastolic blood pressure (79-72 mm Hg; P=0.04), 24-hour ambulatory systolic (130-123 mm Hg; P=0.02) and diastolic blood pressure (67-62 mm Hg; P=0.02), and carotid-femoral pulse wave velocity (12.4-11.0 m/s; P=0.03). Urinary F2-isoprostanes decreased by 31% (209-144 pmol/mmol Cr; P=0.02) despite increased urinary aldosterone excretion. The reduction in urinary F2-isoprostanes closely correlated with the reduction in urinary sodium excretion on the DASH/SRD. In this cohort of HFPEF patients with treated hypertension, the DASH/SRD reduced systemic blood pressure, arterial stiffness, and oxidative stress. These findings are characteristic of salt-sensitive hypertension, a phenotype present in many HFPEF animal models and suggest shared pathophysiological mechanisms linking these 2 conditions. Further dietary modification studies could provide insights into the development and progression of hypertensive HFPEF.

US: immigration court allows Zambian woman's asylum claim based on HIV infection.

On 9 February 2004, the federal Immigration Court ruled that a Zambian woman can stay in the US because her HIV-positive status gives her a well-founded fear of persecution in Zambia. The court found that she would face severe and lethal discrimination in the public health clinics and in the employment sector and that the Zambian government is unwilling or unable to control this persecution.

UK: Court of Appeal orders retrial in UK's first HIV criminal transmission case.

On 5 May 2004, the UK Court of Appeal, Criminal Division, granted Mohammed Dica's appeal against his October 2003 conviction on two counts of causing grievous bodily harm for reckless transmission of HIV. The court ordered a retrial after determining that the trial judge was wrong to not allow the defendant to present information that the victims had known about his condition and had consented to the risk.

Australia: Supreme Court of Western Australia orders retrial in case of man convicted of infecting girlfriend with HIV.

On 3 October 2002, the District Court convicted an HIV-positive man, Houghton, of unlawfully causing grievous bodily harm to his girlfriend for having unprotected vaginal and anal intercourse with her. Houghton had not told her of his HIV status prior to intercourse. He had been aware of his status for some years before meeting the woman, but testified at his trial that he believed that he could not transmit HIV if he did not ejaculate inside the woman. The woman became HIV-positive as a result of the sexual intercourse. The question put before the jury was twofold: whether there had been bodily injury caused by the applicant to the complainant; and whether that injury was of sufficient severity to constitute grievous bodily harm. The jury found Houghton guilty and in doing so made a finding that the transmission of HIV constitutes grievous bodily harm.