Adaptation of SARS-CoV-2 to ACE2H353K mice reveals new spike residues that drive mouse infection.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.

Robotic Versus Laparoscopic Liver Resection in Various Settings: An International Multicenter Propensity Score Matched Study of 10.075 Patients.

OBJECTIVE: To compare the perioperative outcomes of robotic liver surgery (RLS) and laparoscopic liver surgery (LLS) in various settings. BACKGROUND: Clear advantages of RLS over LLS have rarely been demonstrated, and the associated costs of robotic surgery are generally higher than those of laparoscopic surgery. Therefore, the exact role of the robotic approach in minimally invasive liver surgery remains to be defined. METHODS: In this international retrospective cohort study, the outcomes of patients who underwent RLS and LLS for all indications between 2009 and 2021 in 34 hepatobiliary referral centers were compared. Subgroup analyses were performed to compare both approaches across several types of procedures: (1) minor resections in the anterolateral (2, 3, 4b, 5, and 6) or (2) posterosuperior segments (1, 4a, 7, 8), and (3) major resections (≥3 contiguous segments). Propensity score matching was used to mitigate the influence of selection bias. The primary outcome was textbook outcome in liver surgery (TOLS), previously defined as the absence of intraoperative incidents ≥grade 2, postoperative bile leak ≥grade B, severe morbidity, readmission, and 90-day or in-hospital mortality with the presence of an R0 resection margin in case of malignancy. The absence of a prolonged length of stay was added to define TOLS+. RESULTS: Among the 10.075 included patients, 1.507 underwent RLS and 8.568 LLS. After propensity score matching, both groups constituted 1.505 patients. RLS was associated with higher rates of TOLS (78.3% vs 71.8%, P < 0.001) and TOLS+ (55% vs 50.4%, P = 0.026), less Pringle usage (39.1% vs 47.1%, P < 0.001), blood loss (100 vs 200 milliliters, P < 0.001), transfusions (4.9% vs 7.9%, P = 0.003), conversions (2.7% vs 8.8%, P < 0.001), overall morbidity (19.3% vs 25.7%, P < 0.001), and microscopically irradical resection margins (10.1% vs. 13.8%, P = 0.015), and shorter operative times (190 vs 210 minutes, P = 0.015). In the subgroups, RLS tended to have higher TOLS rates, compared with LLS, for minor resections in the posterosuperior segments (n = 431 per group, 75.9% vs 71.2%, P = 0.184) and major resections (n = 321 per group, 72.9% vs 67.5%, P = 0.086), although these differences did not reach statistical significance. CONCLUSIONS: While both produce excellent outcomes, RLS might facilitate slightly higher TOLS rates than LLS.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Type 1 interferon auto-antibodies are elevated in patients with decompensated liver cirrhosis.

Patients with decompensated liver cirrhosis, in particular those classified as Childs-Pugh class C, are at increased risk of severe coronavirus disease-2019 (COVID-19) upon infection with severe acute respiratory coronavirus 2 (SARS-CoV-2). The biological mechanisms underlying this are unknown. We aimed to examine the levels of serum intrinsic antiviral proteins as well as alterations in the innate antiviral immune response in patients with decompensated liver cirrhosis. Serum from 53 SARS-CoV-2 unexposed and unvaccinated individuals, with decompensated liver cirrhosis undergoing assessment for liver transplantation, were screened using SARS-CoV-2 pseudoparticle and SARS-CoV-2 virus assays. The ability of serum to inhibit interferon (IFN) signalling was assessed using a cell-based reporter assay. Severity of liver disease was assessed using two clinical scoring systems, the Child-Pugh class and the MELD-Na score. In the presence of serum from SARS-CoV-2 unexposed patients with decompensated liver cirrhosis there was no association between SARS-CoV-2 pseudoparticle infection or live SARS-CoV-2 virus infection and severity of liver disease. Type I IFNs are a key component of the innate antiviral response. Serum from patients with decompensated liver cirrhosis contained elevated levels of auto-antibodies capable of binding IFN-α2b compared to healthy controls. High MELD-Na scores were associated with the ability of these auto-antibodies to neutralize type I IFN signalling by IFN-α2b but not IFN-ß1a. Our results demonstrate that neutralizing auto-antibodies targeting IFN-α2b are increased in patients with high MELD-Na scores. The presence of neutralizing type I IFN-specific auto-antibodies may increase the likelihood of viral infections, including severe COVID-19, in patients with decompensated liver cirrhosis.

The first few days of a SARS-CoV-2 infection viewed at single-cell resolution.

What transpires soon after inhaling Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the respiratory virus causing Coronavirus Disease 2019 (COVID-19)? Where does infection begin? What are the features of subsequent virus spread? How might host responses quickly contain infection? Two recently published manuscripts have evaluated infection in primary cultures of well-differentiated cells to address these questions and bring more light on the proviral and antiviral components operating during the initial days after SARS-CoV-2 exposure.

Middle East respiratory syndrome coronavirus Spike protein variants exhibit geographic differences in virulence.

Human Middle East respiratory syndrome (MERS) cases were detected primarily in the Middle East before a major outbreak occurred in South Korea in 2015. The Korean outbreak was initiated by a single infected individual, allowing studies of virus evolution in the absence of further MERS-CoV introduction into human populations. In contrast, MERS is primarily a camel disease on the Arabian Peninsula and in Africa, with clinical disease in humans only in the former location. Previous work identified two mutations in the South Korean MERS-CoV, D510G and I529T on the Spike (S) protein, that led to impaired binding to the receptor. However, whether these mutations affected virulence is unknown. To address this question, we constructed isogenic viruses expressing mutations found in the S protein from Korean isolates and showed that isogenic viruses carrying the Korean MERS-CoV mutations, D510G or I529T, were attenuated in mice, resulting in greater survival, less induction of inflammatory cytokines, and less severe lung injury. In contrast, isogenic viruses expressing S proteins from African isolates were nearly fully virulent; other studies showed that West African camel isolates carry mutations in MERS-CoV accessory proteins, which may limit human transmission. These data indicate that following a single-point introduction of the virus, MERS-CoV S protein evolved rapidly in South Korea to adapt to human populations, with consequences on virulence. In contrast, the mutations in S proteins of African isolates did not change virulence, indicating that S protein variation likely does not play a major role in the lack of camel-to-human transmission in Africa.

International Hepato-Pancreato-Billiary Association (IHPBA) registry study on COVID-19 infections in HPB surgery patients.

BACKGROUND: In response to the pandemic, the International Hepato-Pancreato-Biliary Association (IHPBA) developed the IHPBA-COVID Registry to capture data on HPB surgery outcomes in COVID-positive patients prior to mass vaccination programs. The aim was to provide a tool to help members gain a better understanding of the impact of COVID-19 on patient outcomes following HPB surgery worldwide. METHODS: An online registry updated in real time was disseminated to all IHPBA, E-AHPBA, A-HPBA and A-PHPBA members to assess the effects of the pandemic on the outcomes of HPB procedures, perioperative COVID-19 management and other aspects of surgical care. RESULTS: One hundred twenty-five patients from 35 centres in 18 countries were included. Seventy-three (58%) patients were diagnosed with COVID-19 preoperatively. Operative mortality after pancreaticoduodenectomy and major hepatectomy was 28% and 15%, respectively, and 2.5% after cholecystectomy. Postoperative complication rates of pancreatic procedures, hepatic interventions and biliary interventions were respectively 80%, 50% and 37%. Respiratory complication rates were 37%, 31% and 10%, respectively. CONCLUSION: This study reveals a high risk of mortality and complication after HPB surgeries in patient infected with COVID-19. The more extensive the procedure, the higher the risk. Nonetheless, an increased risk was observed across all types of interventions, suggesting that elective HPB surgery should be avoided in COVID positive patients, delaying it at distance from the viral infection.

SARS Coronavirus Redux.

As an atypical pneumonia began to appear in December 2019, Zhou et al. worked with remarkable speed to identify the associated virus, determine its relationship to animal viruses, and evaluate factors conferring infection susceptibility and resistance. These foundational results are being advanced to control the current worldwide human coronavirus epidemic.

Outruling cholangiocarcinoma in patients with primary sclerosing cholangitis wait-listed for liver transplantation: A report on the Irish national experience.

BACKGROUND: The presence of diffuse biliary stricturing in Primary Sclerosing Cholangitis (PSC) makes the diagnosis of early Cholangiocarcinoma (CCA) in this context difficult. A finding of incidental CCA on liver explant is associated with poor oncological outcomes, despite this; there remains no international consensus on how best to outrule CCA in this group ahead of transplantation. The objectives of this study were to report the Irish incidence of incidental CCA in individuals with PSC undergoing liver transplantation, and to critically evaluate the accuracy of diagnostic modalities in outruling CCA in our wait-listed PSC cohort. METHODS: We conducted a retrospective analysis of our prospectively maintained database, which included all PSC patients wait-listed for liver transplant in Ireland. RESULTS: 4.41% of patients (n = 3) were found to have an incidental finding of CCA on liver explant. Despite only being performed in 35.06% of wait-listed PSC patients (n = 27), Endoscopic Retrograde Cholangiopancreatogram (ERCP) with brush cytology was found to be the most effective tool in correctly outruling CCA in this context; associated with a specificity of 96.15%. CONCLUSION: Our findings support a future role for routine surveillance of PSC patients awaiting liver transplantation; however further research is required in order to identify which investigative modalities are of optimal diagnostic utility in this specific context.

Clinical Outcomes After Total Pancreatectomy: A Prospective Multicenter Pan-European Snapshot Study.

OBJECTIVE: To assess outcomes among patients undergoing total pancreatectomy (TP) including predictors for complications and in-hospital mortality. BACKGROUND: Current studies on TP mostly originate from high-volume centers and span long time periods and therefore may not reflect daily practice. METHODS: This prospective pan-European snapshot study included patients who underwent elective (primary or completion) TP in 43 centers in 16 European countries (June 2018-June 2019). Subgroup analysis included cutoff values for annual volume of pancreatoduodenectomies (<60 vs ≥60).Predictors for major complications and in-hospital mortality were assessed in multivariable logistic regression. RESULTS: In total, 277 patients underwent TP, mostly for malignant disease (73%). Major postoperative complications occurred in 70 patients (25%). Median hospital stay was 12 days (IQR 9-18) and 40 patients were readmitted (15%). In-hospital mortality was 5% and 90-day mortality 8%. In the subgroup analysis, in-hospital mortality was lower in patients operated in centers with ≥60 pancreatoduodenectomies compared <60 (4% vs 10%, P = 0.046). In multivariable analysis, annual volume <60 pancreatoduodenectomies (OR 3.78, 95% CI 1.18-12.16, P = 0.026), age (OR 1.07, 95% CI 1.01-1.14, P = 0.046), and estimated blood loss ≥2L (OR 11.89, 95% CI 2.64-53.61, P = 0.001) were associated with in-hospital mortality. ASA ≥3 (OR 2.87, 95% CI 1.56-5.26, P = 0.001) and estimated blood loss ≥2L (OR 3.52, 95% CI 1.25-9.90, P = 0.017) were associated with major complications. CONCLUSION: This pan-European prospective snapshot study found a 5% inhospital mortality after TP. The identified predictors for mortality, including low-volume centers, age, and increased blood loss, may be used to improve outcomes.

Risk of pancreatic ductal adenocarcinoma associated with carriage of BRCA1 and/or BRCA2 mutation: A systematic review and meta-analysis.

Germline BRCA1/2 mutations lead to malfunction of DNA damage repair pathways and predispose to pancreatic ductal adenocarcinoma (PDAC). The aim of this study is to synthesise the available research on this topic. Four studies reporting risk ratio (RR) were included in the final meta-analysis to minimise misrepresenting our results by combining separate risk estimates. Our meta-analysis revealed a statistically significant increased risk of PDAC in BRCA carriers overall (RR: 2.65, 95% confidence interval: 1.43-4.91, p = 0.002).

The role of bariatric surgery in liver transplantation: timing and type.

INTRODUCTION: The rise in obesity worldwide has shifted the indications for liver transplantation (LT), with non-alcoholic steatohepatitis (NASH) being the second most common indication for transplantation. There remains an underestimation of cirrhosis being attributed to NASH. Bariatric surgery (BS) is a reliable solution to overcome obesity and its associated comorbidities. The role of BS in LT has been investigated by different studies; however, the type of BS and timing of LT need further investigation. METHODS: A systemic review examining the role of BS in LT patients was performed. After selection of the studies based on inclusion and exclusion criteria, data extraction was performed by two independent reviewers. Primary outcomes included patient and graft survival. RESULTS: From a total of 2374 articles, five met the prefined criteria. One hundred sixty-two patients had both BS + LT and 1426 underwent LT alone. The percentage of female patients in the BS + LT and LT cohorts was 75% and 35% respectively. The average age in BS + LT and LT cohorts was 43.05 vs. 56.22 years respectively. Patients undergoing BS had comparable outcomes in terms of overall patient survival, graft survival and post-operative morbidity compared to LT alone. When comparing BMI change in patients with prior versus simultaneous BS + LT, no significant difference was found. CONCLUSION: BS and LT patients achieve comparable outcomes to general LT populations. Further studies examining simultaneous BS + LT are needed to answer questions concerning patient selection and timing of surgery.

Pragmatic mAb lead molecule engineering from a developability perspective.

As the number of antibody drugs being approved and marketed increases, our knowledge of what makes potential drug candidates a successful product has increased tremendously. One of the critical parameters that have become clear in the field is the importance of mAb "developability." Efforts are being increasingly focused on simultaneously selecting molecules that exhibit both desirable biological potencies and manufacturability attributes. In the current study mutations to improve the developability profile of a problematic antibody that inconsistently precipitates in a batch scale-dependent fashion using a standard platform purification process are described. Initial bioinformatic analysis showed the molecule has no obvious sequence or structural liabilities that might lead it to precipitate. Subsequent analysis of the molecule revealed the presence of two unusual positively charged mutations on the light chain at the interface of VH and VL domains, which were hypothesized to be the primary contributor to molecule precipitation during process development. To investigate this hypothesis, straightforward reversion to the germline of these residues was carried out. The resulting mutants have improved expression titers and recovered stability within a forced precipitation assay, without any change to biological activity. Given the time pressures of drug development in industry, process optimization of the lead molecule was carried out in parallel to the "retrospective" mutagenesis approach. Bespoke process optimization for large-scale manufacturing was successful. However, we propose that such context-dependent sequence liabilities should be included in the arsenal of in silico developability screening early in development; particularly since this specific issue can be efficiently mitigated without the requirement for extensive screening of lead molecule variants.

Tetraspanins: Architects of Viral Entry and Exit Platforms.

Host factors render cells susceptible to viral infection. One family of susceptibility factors, the tetraspanin proteins, facilitate enveloped virus entry by promoting virus-cell membrane fusion. They also facilitate viral egress from infected cells. In this Gem, we discuss recent insights into how tetraspanins assemble viral entry and exit platforms on cell membranes, and we speculate that tetraspanins contribute to nonviral membrane fusions by similar mechanisms.

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERSMA) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERSMA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERSMA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERSMA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERSMA provide tools to investigate disease causes and develop new therapies.

Hepatopancreatoduodenectomy -a controversial treatment for bile duct and gallbladder cancer from a European perspective.

BACKGROUND: Hepatopancreatoduodenectomy (HPD) is an aggressive operation for treatment of advanced bile duct and gallbladder cancer associated with high perioperative morbidity and mortality, and uncertain oncological benefit in terms of survival. Few reports on HPD from Western centers exist. The purpose of this study was to evaluate safety and efficacy for HPD in European centers. METHOD: Members of the European-African HepatoPancreatoBiliary Association were invited to report all consecutive patients operated with HPD for bile duct or gallbladder cancer between January 2003 and January 2018. The patient and tumor characteristics, perioperative and survival outcomes were analyzed. RESULTS: In total, 66 patients from 19 European centers were included in the analysis. 90-day mortality rate was 17% and 13% for bile duct and gallbladder cancer respectively. All factors predictive of perioperative mortality were patient and disease-specific. The three-year overall survival excluding 90-day mortality was 80% for bile duct and 30% for gallbladder cancer (P = 0.013). In multivariable analysis R0-resection had a significant impact on overall survival. CONCLUSION: HPD, although being associated with substantial perioperative mortality, can offer a survival benefit in patient subgroups with bile duct cancer and gallbladder cancer. To achieve negative resection margins is paramount for an improved survival outcome.

The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases.

Infection by enveloped coronaviruses (CoVs) initiates with viral spike (S) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound S proteins, which prompts S protein-mediated virus-cell membrane fusion. Infection therefore requires close proximity of receptors and proteases. We considered whether tetraspanins, scaffolding proteins known to facilitate CoV infections, hold receptors and proteases together on cell membranes. Using knockout cell lines, we found that the tetraspanin CD9, but not the tetraspanin CD81, formed cell-surface complexes of dipeptidyl peptidase 4 (DPP4), the MERS-CoV receptor, and the type II transmembrane serine protease (TTSP) member TMPRSS2, a CoV-activating protease. This CD9-facilitated condensation of receptors and proteases allowed MERS-CoV pseudoviruses to enter cells rapidly and efficiently. Without CD9, MERS-CoV viruses were not activated by TTSPs, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. Thus, we identified DPP4:CD9:TTSP as the protein complexes necessary for early, efficient MERS-CoV entry. To evaluate the importance of these complexes in an in vivo CoV infection model, we used recombinant Adenovirus 5 (rAd5) vectors to express human DPP4 in mouse lungs, thereby sensitizing the animals to MERS-CoV infection. When the rAd5-hDPP4 vectors co-expressed small RNAs silencing Cd9 or Tmprss2, the animals were significantly less susceptible, indicating that CD9 and TMPRSS2 facilitated robust in vivo MERS-CoV infection of mouse lungs. Furthermore, the S proteins of virulent mouse-adapted MERS-CoVs acquired a CD9-dependent cell entry character, suggesting that CD9 is a selective agent in the evolution of CoV virulence.

Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans from zoonotic sources and causes severe pulmonary disease. Virions require spike (S) glycoproteins for binding to cell receptors and for catalyzing virus-cell membrane fusion. Fusion occurs only after S proteins are cleaved sequentially, first during their secretion through the exocytic organelles of virus-producing cells, and second after virus binding to target-cell receptors. To more precisely determine how sequential proteolysis contributes to CoV infection, we introduced S mutations obstructing the first cleavages. These mutations severely compromised MERS-CoV infection into human lung-derived cells, but had little effect on infection into several other cell types. These cell type-specific requirements for proteolysis correlated with S conformations during cell entry. Without the first cleavages, S proteins resisted cell receptor-induced conformational changes, which restricted the second, fusion-activating cleavages. Consistent with these findings, precleaved MERS viruses used receptor-proximal, cell-surface proteases to effect the second fusion-activating cleavages during cell entry, whereas the more rigid uncleaved MERS viruses trafficked past these cell-surface proteases and into endosomes. Uncleaved viruses were less infectious to human airway epithelial and Calu3 cell cultures because they lacked sufficient endosomal fusion-activating proteases. Thus, by sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.

Neurovirulent Murine Coronavirus JHM.SD Uses Cellular Zinc Metalloproteases for Virus Entry and Cell-Cell Fusion.

The coronavirus (CoV) S protein requires cleavage by host cell proteases to mediate virus-cell and cell-cell fusion. Many strains of the murine coronavirus mouse hepatitis virus (MHV) have distinct, S-dependent organ and tissue tropisms despite using a common receptor, suggesting that they employ different cellular proteases for fusion. In support of this hypothesis, we found that inhibition of endosomal acidification only modestly decreased entry, and overexpression of the cell surface protease TMPRSS2 greatly enhanced entry, of the highly neurovirulent MHV strain JHM.SD relative to their effects on the reference strain, A59. However, TMPRSS2 overexpression decreased MHV structural protein expression, release of infectious particles, and syncytium formation, and endogenous serine protease activity did not contribute greatly to infection. We therefore investigated the importance of other classes of cellular proteases and found that inhibition of matrix metalloproteinase (MMP)- and a disintegrin and metalloprotease (ADAM)-family zinc metalloproteases markedly decreased both entry and cell-cell fusion. Suppression of virus by metalloprotease inhibition varied among tested cell lines and MHV S proteins, suggesting a role for metalloprotease use in strain-dependent tropism. We conclude that zinc metalloproteases must be considered potential contributors to coronavirus fusion.IMPORTANCE The family Coronaviridae includes viruses that cause two emerging diseases of humans, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), as well as a number of important animal pathogens. Because coronaviruses depend on host protease-mediated cleavage of their S proteins for entry, a number of protease inhibitors have been proposed as antiviral agents. However, it is unclear which proteases mediate in vivo infection. For example, SARS-CoV infection of cultured cells depends on endosomal acid pH-dependent proteases rather than on the cell surface acid pH-independent serine protease TMPRSS2, but Zhou et al. (Antiviral Res 116:76-84, 2015, doi:10.1016/j.antiviral.2015.01.011) found that a serine protease inhibitor was more protective than a cathepsin inhibitor in SARS-CoV-infected mice. This paper explores the contributions of endosomal acidification and various proteases to coronavirus infection and identifies an unexpected class of proteases, the matrix metalloproteinase and ADAM families, as potential targets for anticoronavirus therapy.

Heart Failure Home Management Challenges and Reasons for Readmission: a Qualitative Study to Understand the Patient's Perspective.

BACKGROUND: Heart failure patients have high 30-day hospital readmission rates. Interventions designed to prevent readmissions have had mixed success. Understanding heart failure home management through the patient's experience may reframe the readmission "problem" and, ultimately, inform alternative strategies. OBJECTIVE: To understand patient and caregiver challenges to heart failure home management and perceived reasons for readmission. DESIGN: Observational qualitative study. PARTICIPANTS: Heart failure patients were recruited from two hospitals and included those who were hospitalized for heart failure at least twice within 30 days and those who had been recently discharged after their first heart failure admission. APPROACH: Open-ended, semi-structured interviews. Conclusions vetted using focus groups. KEY RESULTS: Semi-structured interviews with 31 patients revealed a combination of physical and socio-emotional influences on patients' home heart failure management. Major themes identified were home management as a struggle between adherence and adaptation, and hospital readmission as a rational choice in response to distressing symptoms. Patients identified uncertainty regarding recommendations, caused by unclear instructions and temporal incongruence between behavior and symptom onset. This uncertainty impaired their competence in making routine management decisions, resulting in a cycle of limit testing and decreasing adherence. Patients reported experiencing hopelessness and frustration in response to perceiving a deteriorating functional status. This led some to a cycle of despair characterized by worsening adherence and negative emotions. As these cycles progressed and distressing symptoms worsened, patients viewed the hospital as the safest place for recovery and not a "negative" outcome. CONCLUSION: Cycles of limit testing and despair represent important patient-centered struggles in managing heart failure. The resulting distress and fear make readmission a rational choice for patients rather than a negative outcome. Interventions (e.g., palliative care) that focus on methods to address these patient-centered factors should be further studied rather than methods to reduce hospital readmissions.