Trajectories of Inflammatory Markers and Post-COVID-19 Cognitive Symptoms: A Secondary Analysis of the CONTAIN COVID-19 Randomized Trial.

BACKGROUND AND OBJECTIVES: Chronic systemic inflammation has been hypothesized to be a mechanistic factor leading to post-acute cognitive dysfunction after COVID-19. However, little data exist evaluating longitudinal inflammatory markers. METHODS: We conducted a secondary analysis of data collected from the CONTAIN randomized trial of convalescent plasma in patients hospitalized for COVID-19, including patients who completed an 18-month assessment of cognitive symptoms and PROMIS Global Health questionnaires. Patients with pre-COVID-19 dementia/cognitive abnormalities were excluded. Trajectories of serum cytokine panels, D-dimer, fibrinogen, C-reactive peptide (CRP), ferritin, lactate dehydrogenase (LDH), and absolute neutrophil counts (ANCs) were evaluated over 18 months using repeated measures and Friedman nonparametric tests. The relationships between the area under the curve (AUC) for each inflammatory marker and 18-month cognitive and global health outcomes were assessed. RESULTS: A total of 279 patients (N = 140 received plasma, N = 139 received placebo) were included. At 18 months, 76/279 (27%) reported cognitive abnormalities and 78/279 (28%) reported fair or poor overall health. PROMIS Global Mental and Physical Health T-scores were 0.5 standard deviations below normal in 24% and 51% of patients, respectively. Inflammatory marker levels declined significantly from hospitalization to 18 months for all markers (IL-2, IL-2R, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, INFγ, TNFα, D-dimer, fibrinogen, ferritin, LDH, CRP, neutrophils; all p < 0.05), with the exception of IL-1ß, which remained stable over time. There were no significant associations between the AUC for any inflammatory marker and 18-month cognitive symptoms, any neurologic symptom, or PROMIS Global Physical or Mental health T-scores. Receipt of convalescent plasma was not associated with any outcome measure. DISCUSSION: At 18 months posthospitalization for COVID-19, cognitive abnormalities were reported in 27% of patients, and below average PROMIS Global Mental and Physical Health scores occurred in 24% and 51%, respectively. However, there were no associations with measured inflammatory markers, which decreased over time.

Inhibiting influenza virus transmission using a broadly acting neuraminidase that targets host sialic acids in the upper respiratory tract.

The ongoing transmission of influenza A viruses (IAV) for the past century continues to be a burden to humans. IAV binds terminal sialic acids (SA) of sugar molecules present within the upper respiratory tract (URT) in order to successfully infect hosts. The two most common SA structures that are important for IAV infection are those with α2,3- and α2,6-linkages. While mice were once considered to be an unsuitable system for studying IAV transmission due to their lack of α2,6-SA in the trachea, we have successfully demonstrated that IAV transmission in infant mice is remarkably efficient. This finding led us to re-evaluate the SA composition of the URT of mice using in situ immunofluorescence and examine its in vivo contribution to transmission for the first time. We demonstrate that mice express both α2,3- and α2,6-SA in the URT and that the difference in expression between infants and adults contributes to the variable transmission efficiencies observed. Furthermore, selectively blocking α2,3-SA or α2,6-SA within the URT of infant mice using lectins was necessary but insufficient at inhibiting transmission, and simultaneous blockade of both receptors was crucial in achieving the desired inhibitory effect. By employing a broadly acting neuraminidase to indiscriminately remove both SA moieties in vivo, we effectively suppressed viral shedding and halted the transmission of different strains of influenza viruses. These results emphasize the utility of the infant mouse model for studying IAV transmission and strongly indicate that broadly targeting host SA is an effective approach that inhibits IAV contagion.IMPORTANCEInfluenza virus transmission studies have historically focused on viral mutations that alter hemagglutinin binding to sialic acid (SA) receptors in vitro. However, SA binding preference does not fully account for the complexities of influenza A virus transmission in humans. Our previous findings reveal that viruses that are known to bind α2,6-SA in vitro have different transmission kinetics in vivo, suggesting that diverse SA interactions may occur during their life cycle. In this study, we examine the role of host SA on viral replication, shedding, and transmission in vivo. We highlight the critical role of SA presence during virus shedding, such that attachment to SA during virion egress is equally important as detachment from SA during virion release. These insights support the potential of broadly acting neuraminidases as therapeutic agents capable of restraining viral transmission in vivo. Our study unveils intricate virus-host interactions during shedding, highlighting the necessity to develop innovative strategies to effectively target transmission.

COVID-19 Convalescent Plasma Therapy: Long-term Implications.

Background: The long-term effect of coronavirus disease 2019 (COVID-19) acute treatments on postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC) is unknown. The CONTAIN-Extend study explores the long-term impact of COVID-19 convalescent plasma (CCP) therapy on postacute sequelae of SARS-CoV-2 infection (PASC) symptoms and general health 18 months following hospitalization. Methods: The CONTAIN-Extend study examined 281 participants from the original CONTAIN COVID-19 trial (CONTAIN-RCT, NCT04364737) at 18 months post-hospitalization for acute COVID-19. Symptom surveys, global health assessments, and biospecimen collection were performed from November 2021 to October 2022. Multivariable logistic and linear regression estimated associations between the randomization arms and self-reported symptoms and Patient-Reported Outcomes Measurement Information System (PROMIS) scores and adjusted for covariables, including age, sex, race/ethnicity, disease severity, and CONTAIN enrollment quarter and sites. Results: There were no differences in symptoms or PROMIS scores between CCP and placebo (adjusted odds ratio [aOR] of general symptoms, 0.95; 95% CI, 0.54-1.67). However, females (aOR, 3.01; 95% CI, 1.73-5.34), those 45-64 years (aOR, 2.55; 95% CI, 1.14-6.23), and April-June 2020 enrollees (aOR, 2.39; 95% CI, 1.10-5.19) were more likely to report general symptoms and have poorer PROMIS physical health scores than their respective reference groups. Hispanic participants (difference, -3.05; 95% CI, -5.82 to -0.27) and Black participants (-4.48; 95% CI, -7.94 to -1.02) had poorer PROMIS physical health than White participants. Conclusions: CCP demonstrated no lasting effect on PASC symptoms or overall health in comparison to the placebo. This study underscores the significance of demographic factors, including sex, age, and timing of acute infection, in influencing symptom reporting 18 months after acute hypoxic COVID-19 hospitalization.

Selective adaptation of SARS-CoV-2 Omicron under booster vaccine pressure: a multicentre observational study.

BACKGROUND: High rates of vaccination and natural infection drive immunity and redirect selective viral adaptation. Updated boosters are installed to cope with drifted viruses, yet data on adaptive evolution under increasing immune pressure in a real-world situation are lacking. METHODS: Cross-sectional study to characterise SARS-CoV-2 mutational dynamics and selective adaptation over >1 year in relation to vaccine status, viral phylogenetics, and associated clinical and demographic variables. FINDINGS: The study of >5400 SARS-CoV-2 infections between July 2021 and August 2022 in metropolitan New York portrayed the evolutionary transition from Delta to Omicron BA.1-BA.5 variants. Booster vaccinations were implemented during the Delta wave, yet booster breakthrough infections and SARS-CoV-2 re-infections were almost exclusive to Omicron. In adjusted logistic regression analyses, BA.1, BA.2, and BA.5 had a significant growth advantage over co-occurring lineages in the boosted population, unlike BA.2.12.1 or BA.4. Selection pressure by booster shots translated into diffuse adaptive evolution in Delta spike, contrasting with strong, receptor-binding motif-focused adaptive evolution in BA.2-BA.5 spike (Fisher Exact tests; non-synonymous/synonymous mutation rates per site). Convergent evolution has become common in Omicron, engaging spike positions crucial for immune escape, receptor binding, or cleavage. INTERPRETATION: Booster shots are required to cope with gaps in immunity. Their discriminative immune pressure contributes to their effectiveness but also requires monitoring of selective viral adaptation processes. Omicron BA.2 and BA.5 had a selective advantage under booster vaccination pressure, contributing to the evolution of BA.2 and BA.5 sublineages and recombinant forms that predominate in 2023. FUNDING: The study was supported by NYU institutional funds and partly by the Cancer Center Support Grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center.

Targeting host sialic acids in the upper respiratory tract with a broadly-acting neuraminidase to inhibit influenza virus transmission.

The ongoing transmission of influenza A viruses (IAV) for the past century continues to be a burden to humans. IAV binds terminal sialic acids (SA) of sugar molecules present within the upper respiratory tract (URT) in order to successfully infect hosts. The two most common SA structures that are important for IAV infection are those with α2,3- and α2,6-linkages. While mice were once considered to be an unsuitable system for studying IAV transmission due to their lack of α2,6-SA in the trachea, we have successfully demonstrated that IAV transmission in infant mice is remarkably efficient. This finding led us to reevaluate the SA composition of the URT of mice using in situ immunofluorescence and examine its in vivo contribution to transmission for the first time. We demonstrate that mice express both α2,3- and α2,6-SA in the URT and that the difference in expression between infants and adults contribute to the variable transmission efficiencies observed. Furthermore, selectively blocking α2,3-SA or α2,6-SA within the URT of infant mice using lectins was necessary but insufficient at inhibiting transmission, and simultaneous blockade of both receptors was crucial in achieving the desired inhibitory effect. By employing a broadly-acting neuraminidase (ba-NA) to indiscriminately remove both SA moieties in vivo, we effectively suppressed viral shedding and halted the transmission of different strains of influenza viruses. These results emphasize the utility of the infant mouse model for studying IAV transmission, and strongly indicate that broadly targeting host SA is an effective approach that inhibits IAV contagion.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.

Delta-Omicron recombinant escapes therapeutic antibody neutralization.

The emergence of recombinant viruses is a threat to public health, as recombination may integrate variant-specific features that together result in escape from treatment or immunity. The selective advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. We identified a Delta-Omicron (AY.45-BA.1) recombinant in an immunosuppressed transplant recipient treated with monoclonal antibody Sotrovimab. The single recombination breakpoint is located in the spike N-terminal domain adjacent to the Sotrovimab binding site. While Delta and BA.1 are sensitive to Sotrovimab neutralization, the Delta-Omicron recombinant is highly resistant. To our knowledge, this is the first described instance of recombination between circulating SARS-CoV-2 variants as a functional mechanism of resistance to treatment and immune escape.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets 1, 2 . Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2 3 . Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein in this context.

Delta-Omicron recombinant escapes therapeutic antibody neutralization.

Background: The emergence of recombinant viruses is a threat to public health. Recombination of viral variants may combine variant-specific features that together catalyze viral escape from treatment or immunity. The selective advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. Methods: Multi-method amplicon and metagenomic sequencing of a clinical swab and the in vitro grown virus allowed for high-confidence detection of a novel recombinant variant. Mutational, phylogeographic, and structural analyses determined features of the recombinant genome and spike protein. Neutralization assays using infectious as well as pseudotyped viruses and point mutants thereof defined the recombinant's sensitivity to a panel of monoclonal antibodies and sera from vaccinated and/or convalescent individuals. Results: A novel Delta-Omicron SARS-CoV-2 recombinant was identified in an unvaccinated, immunosuppressed kidney transplant recipient treated with monoclonal antibody Sotrovimab. The recombination breakpoint is located in the spike N-terminal domain, adjacent to the Sotrovimab quaternary binding site, and results in a 5'-Delta AY.45 and a 3'-Omicron BA.1 mosaic spike protein. Delta and BA.1 are sensitive to Sotrovimab neutralization, whereas the Delta-Omicron recombinant is highly resistant to Sotrovimab, both with and without the RBD resistance mutation E340D. Conclusions: Recombination between circulating SARS-CoV-2 variants can functionally contribute to immune escape. It is critical to validate phenotypes of mosaic viruses and monitor immunosuppressed COVID-19 patients treated with monoclonal antibodies for the selection of recombinant and immune escape variants. (Funded by NYU, the National Institutes of Health, and others).

Association of Convalescent Plasma Treatment With Clinical Status in Patients Hospitalized With COVID-19: A Meta-analysis.

Importance: COVID-19 convalescent plasma (CCP) is a potentially beneficial treatment for COVID-19 that requires rigorous testing. Objective: To compile individual patient data from randomized clinical trials of CCP and to monitor the data until completion or until accumulated evidence enables reliable conclusions regarding the clinical outcomes associated with CCP. Data Sources: From May to August 2020, a systematic search was performed for trials of CCP in the literature, clinical trial registry sites, and medRxiv. Domain experts at local, national, and international organizations were consulted regularly. Study Selection: Eligible trials enrolled hospitalized patients with confirmed COVID-19, not receiving mechanical ventilation, and randomized them to CCP or control. The administered CCP was required to have measurable antibodies assessed locally. Data Extraction and Synthesis: A minimal data set was submitted regularly via a secure portal, analyzed using a prespecified bayesian statistical plan, and reviewed frequently by a collective data and safety monitoring board. Main Outcomes and Measures: Prespecified coprimary end points-the World Health Organization (WHO) 11-point ordinal scale analyzed using a proportional odds model and a binary indicator of WHO score of 7 or higher capturing the most severe outcomes including mechanical ventilation through death and analyzed using a logistic model-were assessed clinically at 14 days after randomization. Results: Eight international trials collectively enrolled 2369 participants (1138 randomized to control and 1231 randomized to CCP). A total of 2341 participants (median [IQR] age, 60 [50-72] years; 845 women [35.7%]) had primary outcome data as of April 2021. The median (IQR) of the ordinal WHO scale was 3 (3-6); the cumulative OR was 0.94 (95% credible interval [CrI], 0.74-1.19; posterior probability of OR <1 of 71%). A total of 352 patients (15%) had WHO score greater than or equal to 7; the OR was 0.94 (95% CrI, 0.69-1.30; posterior probability of OR <1 of 65%). Adjusted for baseline covariates, the ORs for mortality were 0.88 at day 14 (95% CrI, 0.61-1.26; posterior probability of OR <1 of 77%) and 0.85 at day 28 (95% CrI, 0.62-1.18; posterior probability of OR <1 of 84%). Heterogeneity of treatment effect sizes was observed across an array of baseline characteristics. Conclusions and Relevance: This meta-analysis found no association of CCP with better clinical outcomes for the typical patient. These findings suggest that real-time individual patient data pooling and meta-analysis during a pandemic are feasible, offering a model for future research and providing a rich data resource.

Development and Validation of a Treatment Benefit Index to Identify Hospitalized Patients With COVID-19 Who May Benefit From Convalescent Plasma.

Importance: Identifying which patients with COVID-19 are likely to benefit from COVID-19 convalescent plasma (CCP) treatment may have a large public health impact. Objective: To develop an index for predicting the expected relative treatment benefit from CCP compared with treatment without CCP for patients hospitalized for COVID-19 using patients' baseline characteristics. Design, Setting, and Participants: This prognostic study used data from the COMPILE study, ie, a meta-analysis of pooled individual patient data from 8 randomized clinical trials (RCTs) evaluating CCP vs control in adults hospitalized for COVID-19 who were not receiving mechanical ventilation at randomization. A combination of baseline characteristics, termed the treatment benefit index (TBI), was developed based on 2287 patients in COMPILE using a proportional odds model, with baseline characteristics selected via cross-validation. The TBI was externally validated on 4 external data sets: the Expanded Access Program (1896 participants), a study conducted under Emergency Use Authorization (210 participants), and 2 RCTs (with 80 and 309 participants). Exposure: Receipt of CCP. Main Outcomes and Measures: World Health Organization (WHO) 11-point ordinal COVID-19 clinical status scale and 2 derivatives of it (ie, WHO score of 7-10, indicating mechanical ventilation to death, and WHO score of 10, indicating death) at day 14 and day 28 after randomization. Day 14 WHO 11-point ordinal scale was used as the primary outcome to develop the TBI. Results: A total of 2287 patients were included in the derivation cohort, with a mean (SD) age of 60.3 (15.2) years and 815 (35.6%) women. The TBI provided a continuous gradation of benefit, and, for clinical utility, it was operationalized into groups of expected large clinical benefit (B1; 629 participants in the derivation cohort [27.5%]), moderate benefit (B2; 953 [41.7%]), and potential harm or no benefit (B3; 705 [30.8%]). Patients with preexisting conditions (diabetes, cardiovascular and pulmonary diseases), with blood type A or AB, and at an early COVID-19 stage (low baseline WHO scores) were expected to benefit most, while those without preexisting conditions and at more advanced stages of COVID-19 could potentially be harmed. In the derivation cohort, odds ratios for worse outcome, where smaller odds ratios indicate larger benefit from CCP, were 0.69 (95% credible interval [CrI], 0.48-1.06) for B1, 0.82 (95% CrI, 0.61-1.11) for B2, and 1.58 (95% CrI, 1.14-2.17) for B3. Testing on 4 external datasets supported the validation of the derived TBIs. Conclusions and Relevance: The findings of this study suggest that the CCP TBI is a simple tool that can quantify the relative benefit from CCP treatment for an individual patient hospitalized with COVID-19 that can be used to guide treatment recommendations. The TBI precision medicine approach could be especially helpful in a pandemic.

Efficacy and Safety of COVID-19 Convalescent Plasma in Hospitalized Patients: A Randomized Clinical Trial.

Importance: There is clinical equipoise for COVID-19 convalescent plasma (CCP) use in patients hospitalized with COVID-19. Objective: To determine the safety and efficacy of CCP compared with placebo in hospitalized patients with COVID-19 receiving noninvasive supplemental oxygen. Design, Setting, and Participants: CONTAIN COVID-19, a randomized, double-blind, placebo-controlled trial of CCP in hospitalized adults with COVID-19, was conducted at 21 US hospitals from April 17, 2020, to March 15, 2021. The trial enrolled 941 participants who were hospitalized for 3 or less days or presented 7 or less days after symptom onset and required noninvasive oxygen supplementation. Interventions: A unit of approximately 250 mL of CCP or equivalent volume of placebo (normal saline). Main Outcomes and Measures: The primary outcome was participant scores on the 11-point World Health Organization (WHO) Ordinal Scale for Clinical Improvement on day 14 after randomization; the secondary outcome was WHO scores determined on day 28. Subgroups were analyzed with respect to age, baseline WHO score, concomitant medications, symptom duration, CCP SARS-CoV-2 titer, baseline SARS-CoV-2 serostatus, and enrollment quarter. Outcomes were analyzed using a bayesian proportional cumulative odds model. Efficacy of CCP was defined as a cumulative adjusted odds ratio (cOR) less than 1 and a clinically meaningful effect as cOR less than 0.8. Results: Of 941 participants randomized (473 to placebo and 468 to CCP), 556 were men (59.1%); median age was 63 years (IQR, 52-73); 373 (39.6%) were Hispanic and 132 (14.0%) were non-Hispanic Black. The cOR for the primary outcome adjusted for site, baseline risk, WHO score, age, sex, and symptom duration was 0.94 (95% credible interval [CrI], 0.75-1.18) with posterior probability (P[cOR<1] = 72%); the cOR for the secondary adjusted outcome was 0.92 (95% CrI, 0.74-1.16; P[cOR<1] = 76%). Exploratory subgroup analyses suggested heterogeneity of treatment effect: at day 28, cORs were 0.72 (95% CrI, 0.46-1.13; P[cOR<1] = 93%) for participants enrolled in April-June 2020 and 0.65 (95% CrI, 0.41 to 1.02; P[cOR<1] = 97%) for those not receiving remdesivir and not receiving corticosteroids at randomization. Median CCP SARS-CoV-2 neutralizing titer used in April to June 2020 was 1:175 (IQR, 76-379). Any adverse events (excluding transfusion reactions) were reported for 39 (8.2%) placebo recipients and 44 (9.4%) CCP recipients (P = .57). Transfusion reactions occurred in 2 (0.4) placebo recipients and 8 (1.7) CCP recipients (P = .06). Conclusions and Relevance: In this trial, CCP did not meet the prespecified primary and secondary outcomes for CCP efficacy. However, high-titer CCP may have benefited participants early in the pandemic when remdesivir and corticosteroids were not in use. Trial Registration: ClinicalTrials.gov Identifier: NCT04364737.

Type I Interferon Signaling Is a Common Factor Driving Streptococcus pneumoniae and Influenza A Virus Shedding and Transmission.

The dynamics underlying respiratory contagion (the transmission of infectious agents from the airways) are poorly understood. We investigated host factors involved in the transmission of the leading respiratory pathogen Streptococcus pneumoniae Using an infant mouse model, we examined whether S. pneumoniae triggers inflammatory pathways shared by influenza A virus (IAV) to promote nasal secretions and shedding from the upper respiratory tract to facilitate transit to new hosts. Here, we show that amplification of the type I interferon (IFN-I) response is a critical host factor in this process, as shedding and transmission by both IAV and S. pneumoniae were decreased in pups lacking the common IFN-I receptor (Ifnar1-/- mice). Additionally, providing exogenous recombinant IFN-I to S. pneumoniae-infected pups was sufficient to increase bacterial shedding. The expression of IFN-stimulated genes (ISGs) was upregulated in S. pneumoniae-infected wild-type (WT) but not Ifnar1-/- mice, including genes involved in mucin type O-glycan biosynthesis; this correlated with an increase in secretions in S. pneumoniae- and IAV-infected WT compared to Ifnar1-/- pups. S. pneumoniae stimulation of ISGs was largely dependent on its pore-forming toxin, pneumolysin, and coinfection with IAV and S. pneumoniae resulted in synergistic increases in ISG expression. We conclude that the induction of IFN-I signaling appears to be a common factor driving viral and bacterial respiratory contagion.IMPORTANCE Respiratory tract infections are a leading cause of childhood mortality and, globally, Streptococcus pneumoniae is the leading cause of mortality due to pneumonia. Transmission of S. pneumoniae primarily occurs through direct contact with respiratory secretions, although the host and bacterial factors underlying transmission are poorly understood. We examined transmission dynamics of S. pneumoniae in an infant mouse model and here show that S. pneumoniae colonization of the upper respiratory tract stimulates host inflammatory pathways commonly associated with viral infections. Amplification of this response was shown to be a critical host factor driving shedding and transmission of both S. pneumoniae and influenza A virus, with infection stimulating expression of a wide variety of genes, including those involved in the biosynthesis of mucin, a major component of respiratory secretions. Our findings suggest a mechanism facilitating S. pneumoniae contagion that is shared by viral infection.

Human host factors required for influenza virus replication.

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.

Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice.

The influenza virus PB1-F2 protein is a novel protein previously shown to be involved in induction of cell death. Here we characterize the expression and the function of the protein within the context of influenza viral infection in tissue culture and a mouse model. We show that the C-terminal region of the protein can be expressed from a downstream initiation codon and is capable of interaction with the full-length protein. Using this knowledge, we generated influenza viruses knocked out for the expression of PB1-F2 protein and its downstream truncation products. Knocking out the PB1-F2 protein had no effect on viral replication in tissue culture but diminished virus pathogenicity and mortality in mice. The viruses replicated to similar levels in mouse lungs by day 3 postinfection, suggesting that the knockout did not impair viral replication. However, while the PB1-F2 knockout viruses were cleared after day 5, the wild-type viruses were detectable in mouse lungs until day 7, implying that expression of PB1-F2 resulted in delayed clearance of the viruses by the host immune system. Based on our findings and on the fact that the PB1 genomic segment was always newly introduced into some pandemic influenza viruses of the last century, we speculate that the PB1-F2 protein plays an important role in pathogenesis of influenza virus infection and may be an important contributor to pathogenicity of pandemic influenza viruses.