Investigation of air dispersal during a rhinovirus outbreak in a pediatric intensive care unit.

BACKGROUND: While airborne transmission of rhinovirus is recognized in indoor settings, its role in hospital transmission remains unclear. METHODS: We investigated an outbreak of rhinovirus in a pediatric intensive care unit (PICU) to assess air dispersal. We collected clinical, environmental, and air samples, and staff's surgical masks for viral load and phylogenetic analysis. Hand hygiene compliance and the number of air changes per hour in the PICU were measured. A case-control analysis was performed to identify nosocomial rhinovirus risk factors. RESULTS: Between March 31, 2023, and April 2, 2023, three patients acquired rhinovirus in a cubicle (air changes per hour: 14) of 12-bed PICU. A portable air-cleaning unit was placed promptly. Air samples (72,000 L in 6 hours) from the cohort area, and outer surfaces of staff's masks (n = 8), were rhinovirus RNA-negative. Hand hygiene compliance showed no significant differences (31/34, 91.2% vs 33/37, 89.2%, P = 1) before and during outbreak. Only 1 environmental sample (3.8%) was positive (1.86 × 103 copies/mL). Case-control and next-generation sequencing analysis implicated an infected staff member as the source. CONCLUSIONS: Our findings suggest that air dispersal of rhinovirus was not documented in the well-ventilated PICU during the outbreak. Further research is needed to better understand the dynamics of rhinovirus transmission in health care settings.

Corrigendum: Co-stimulation with TLR7 agonist imiquimod and inactivated Influenza virus particles promotes mouse B cell activation, differentiation, and accelerated antigen specific antibody production.

[This corrects the article DOI: 10.3389/fimmu.2018.02370.].

Factors associated with death due to severe acute respiratory syndrome caused by influenza: Brazilian population study.

INTRODUCTION: Influenza infection is characterized by acute viral infection of high transmissibility. Worsening of the case can lead to the need for hospitalization, severe acute respiratory syndrome (SARS) and even death. METHOD: This is a cross-sectional population-based study that used secondary database from the Brazilian Influenza Epidemiological Surveillance Information System. Only cases of adults with diagnosis of influenza by RT-PCR and case evolution recorded were included. RESULTS: We identified 2273 adults with SARS by influenza, 343 of which had death as an outcome. The main risk factors for death were lack of hospitalization, not having cough and age, both with p < 0.001. In addition, without asthma, having black skin color, not receiving flu vaccine, having brown skin color and not having a sore throat (p ≤ 0.005) were risk factors too. CONCLUSION: Factors associated with death due to SARS caused by influenza in Brazil, risk factors and protective factors to death were identified. It was evident that those who did not receive the flu vaccine presented twice the risk of unfavorable outcome, reinforcing the need to stimulate adherence to vaccination adhering and propose changes in public policies to make influenza vaccines available to the entire population, in order to prevent severe cases and unfavorable outcomes.

Medium-term outcomes of myocarditis and pericarditis following BNT162b2 vaccination among adolescents in Hong Kong.

In this study, we examined the clinical and electrophysiological outcomes of adolescents in Hong Kong who developed myocarditis or pericarditis following BNT162b2 vaccination for COVID-19, and followed-up for 60-180 days after their initial diagnosis. Clinical assessments included electrocardiogram (ECG) and echocardiogram at the initial admission and follow-up were compared. Treadmill testing was also performed in some cases. Between 14 June 2021 and 16 February 2022, 53 subjects were approached to participate in this follow-up study, of which 28 patients were followed up for >60 days with a median follow-up period of 100 days (range, 61-178 days) and were included in this study. On admission, 23 patients had ECG abnormalities but no high-grade atrioventricular block. Six patients had echocardiogram abnormalities, including reduced contractility, small rim pericardial effusions, and hyperechoic ventricular walls. All patients achieved complete recovery on follow-up. After discharge, 10 patients (35.7%) reported symptoms, including occasional chest pain, shortness of breath, reduced exercise tolerance, and recurrent vasovagal near-syncope. At follow-up, assessments, including ECGs, were almost all normal. Among the three patients with possible ECG abnormalities, all their echocardiograms or treadmill testings were normal. Sixteen patients (57.1%) underwent treadmill testing at a median of 117 days post-admission, which were also normal. However, at follow-up, there was a significant mean bodyweight increase of 1.81 kg (95%CI 0.47-3.1 kg, p = 0.01), possibly due to exercise restriction. In conclusion, most adolescents experiencing myocarditis and pericarditis following BNT162b2 vaccination achieved complete recovery. Some patients developed non-specific persistent symptoms, and bodyweight changes shall be monitored.

Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2.

The B.1.1.529/Omicron variant of SARS-CoV-2 was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 vaccines and antibody therapies4. This concern is amplified by the findings of our study. Here we found that B.1.1.529 is markedly resistant to neutralization by serum not only from patients who recovered from COVID-19, but also from individuals who were vaccinated with one of the four widely used COVID-19 vaccines. Even serum from individuals who were vaccinated and received a booster dose of mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies against all known epitope clusters on the spike protein, we noted that the activity of 17 out of the 19 antibodies tested were either abolished or impaired, including ones that are currently authorized or approved for use in patients. Moreover, we also identified four new spike mutations (S371L, N440K, G446S and Q493R) that confer greater antibody resistance on B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.

Comparative evaluation of a dual-target real-time RT-PCR assay for COVID-19 diagnosis and assessment of performance in pooled saliva and nasopharyngeal swab samples.

OBJECTIVES: Sensitive molecular diagnostic assays are essential for COVID-19 diagnosis. We evaluated the Hecin Scientific SARS-CoV-2 nucleic acid test kit, a dual-target real-time RT-PCR assay targeting the SARS-CoV-2 N and ORF1ab genes. METHODS: The Hecin test kit's diagnostic performance in detecting SARS-CoV-2 RNA was compared to the LightMix Modular SARS and Wuhan CoV E-gene kit (TIB Molbiol) and an in-house single-tube nested real-time RT-PCR using 296 clinical specimens, 11 proficiency testing samples, and 30 low-positive deep throat saliva and nasopharyngeal swab (NPS) samples pooled into negative samples in ratios of 1:5, 1:10, and 1:30. RESULTS: The limit-of-detection of the Hecin test kit was around 500 dC/mL for the N and ORF1ab targets. Sensitivity and specificity of the Hecin test kit were 98.1% (95% CI: 93.4-99.8%) and 100% (98.1-100%), respectively, when measured against the reference method. The Hecin test kit showed fair sensitivity (80%) in low-positive NPS samples pooled in ratios of 1:5 and 1:10. Its performance in pooled samples could be dramatically improved by adjusting the assay Ct cutoff. CONCLUSION: The Hecin test kit enables sensitive and specific detection of SARS-CoV-2 in clinical samples and pooled samples.

Clinical Characteristics and Transmission of COVID-19 in Children and Youths During 3 Waves of Outbreaks in Hong Kong.

Importance: Schools were closed intermittently across Hong Kong to control the COVID-19 outbreak, which led to significant physical and psychosocial problems among children and youths. Objective: To compare the clinical characteristics and sources of infection among children and youths with COVID-19 during the 3 waves of outbreaks in Hong Kong in 2020. Design, Setting, and Participants: This cross-sectional study involved children and youths aged 18 years or younger with COVID-19 in the 3 waves of outbreaks from January 23 through December 2, 2020. Data were analyzed from December 2020 through January 2021. Main Outcomes and Measures: Demographic characteristics, travel and contact histories, lengths of hospital stay, and symptoms were captured through the central electronic database. Individuals who were infected without recent international travel were defined as having domestic infections. Results: Among 397 children and youths confirmed with COVID-19 infections, the mean (SD) age was 9.95 (5.34) years, 220 individuals (55.4%) were male, and 154 individuals (38.8%) were asymptomatic. There were significantly more individuals who were infected without symptoms in the second wave (59 of 118 individuals [50.0%]) and third wave (94 of 265 individuals [35.5%]) than in the first wave (1 of 14 individuals [7.1%]) (P = .001). Significantly fewer individuals who were infected in the second and third waves, compared with the first wave, had fever (first wave: 10 individuals [71.4%]; second wave: 22 individuals [18.5%]; third wave: 98 individuals [37.0%]; P < .001) or cough (first wave: 6 individuals [42.9%]; second wave: 15 individuals [12.7%]; third wave: 52 individuals [19.6%]; P = .02). Among all individuals, 394 individuals (99.2%) had mild illness. One patient developed chilblains (ie, COVID toes), 1 patient developed multisystem inflammatory syndrome in children, and 1 patient developed post-COVID-19 autoimmune hemolytic anemia. In all 3 waves, 204 patients with COVID-19 (51.4%) had domestic infections. Among these individuals, 186 (91.2%) reported having a contact history with another individual with COVID-19, of which most (183 individuals [90.0%]) were family members. In the third wave, 18 individuals with domestic infections had unknown contact histories. Three schoolmates were confirmed with COVID-19 on the same day and were reported to be close contacts. Conclusions and Relevance: This cross-sectional study found that nearly all children and youths with COVID-19 in Hong Kong had mild illness. These findings suggest that household transmission was the main source of infection for children and youths with domestic infections and that the risk of being infected at school was small.

Mining of linear B cell epitopes of SARS-CoV-2 ORF8 protein from COVID-19 patients.

Given the on-going SARS-CoV-2 pandemic, identification of immunogenic targets against the viral protein will provide crucial advances towards the development of sensitive diagnostic tools and vaccination strategies. Our previous study has found that ORF8 protein of SARS-CoV-2 is highly immunogenic and shows high sensitivity in identifying COVID-19 disease. In this study, by employing overlapping linear peptides, we characterized the IgG immunodominant regions on SARS-CoV-2 ORF8 protein that are seropositive in the sera from SARS-CoV-2-infected patients. The major immunogenic epitopes are localized at (1) N-termini alpha helix, (2) the resides spanning beta 2 and 3 sheets, and (3) the loop between beta 4 and 5 sheets. Additionally, hamster model infected by SARS-CoV-2 further validates the seropositivity of the linear epitopes in vivo, demonstrating a potential application of the linear peptide-based immunization strategy. Taken together, identification and validation of these B-cell linear epitopes will provide insights into the design of serological diagnostics and peptide-based vaccination approach against this pandemic virus of high priority.

Cross-linking peptide and repurposed drugs inhibit both entry pathways of SARS-CoV-2.

Up to date, effective antivirals have not been widely available for treating COVID-19. In this study, we identify a dual-functional cross-linking peptide 8P9R which can inhibit the two entry pathways (endocytic pathway and TMPRSS2-mediated surface pathway) of SARS-CoV-2 in cells. The endosomal acidification inhibitors (8P9R and chloroquine) can synergistically enhance the activity of arbidol, a spike-ACE2 fusion inhibitor, against SARS-CoV-2 and SARS-CoV in cells. In vivo studies indicate that 8P9R or the combination of repurposed drugs (umifenovir also known as arbidol, chloroquine and camostat which is a TMPRSS2 inhibitor), simultaneously interfering with the two entry pathways of coronaviruses, can significantly suppress SARS-CoV-2 replication in hamsters and SARS-CoV in mice. Here, we use drug combination (arbidol, chloroquine, and camostat) and a dual-functional 8P9R to demonstrate that blocking the two entry pathways of coronavirus can be a promising and achievable approach for inhibiting SARS-CoV-2 replication in vivo. Cocktail therapy of these drug combinations should be considered in treatment trials for COVID-19.

Saliva viral load better correlates with clinical and immunological profiles in children with coronavirus disease 2019.

BACKGROUND: Pediatric COVID-19 studies exploring the relationships between NPS and saliva viral loads, clinical and immunological profiles are lacking. METHODS: Demographics, immunological profiles, nasopharyngeal swab (NPS), and saliva samples collected on admission, and hospital length of stay (LOS) were assessed in children below 18 years with COVID-19. FINDINGS: 91 patients were included between March and August 20 20. NPS and saliva viral loads were correlated (r = 0.315, p = 0.01). Symptomatic patients had significantly higher NPS and saliva viral loads than asymptomatic patients. Serial NPS and saliva viral load measurements showed that the log10 NPS (r = -0.532, p < 0.001) and saliva (r = -0.417, p < 0.001) viral loads for all patients were inversely correlated with the days from symptom onset with statistical significance. Patients with cough, sputum, and headache had significantly higher saliva, but not NPS, viral loads. Higher saliva, but not NPS, viral loads were associated with total lymphopenia, CD3 and CD4 lymphopenia (all p < 0.05), and were inversely correlated with total lymphocyte (r = -0.43), CD3 (r = -0.55), CD4 (r = -0.60), CD8 (r = -0.41), B (r = -0.482), and NK (r = -0.416) lymphocyte counts (all p < 0.05). INTERPRETATION: Saliva viral loads on admission in children correlated better with clinical and immunological profiles than NPS.

Assessment of SARS-CoV-2 Immunity in Convalescent Children and Adolescents.

Persistence of protective immunity for SARS-CoV-2 is important against reinfection. Knowledge on SARS-CoV-2 immunity in pediatric patients is currently lacking. We opted to assess the SARS-CoV-2 adaptive immunity in recovered children and adolescents, addressing the pediatrics specific immunity towards COVID-19. Two independent assays were performed to investigate humoral and cellular immunological memory in pediatric convalescent COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell responses were identified and quantified in recovered children and adolescents. SARS-CoV-2-specific RBD IgG detected in recovered patients had a half-life of 121.6 days and estimated duration of 7.9 months compared with baseline levels in controls. The specific T cell response was shown to be independent of days after diagnosis. Both CD4+ and CD8+ T cells showed robust responses not only to spike (S) peptides (a main target of vaccine platforms) but were also similarly activated when stimulated by membrane (M) and nuclear (N) peptides. Importantly, we found the differences in the adaptive responses were correlated with the age of the recovered patients. The CD4+ T cell response to SARS-CoV-2 S peptide in children aged <12 years correlated with higher SARS-CoV-2 RBD IgG levels, suggesting the importance of a T cell-dependent humoral response in younger children under 12 years. Both cellular and humoral immunity against SARS-CoV-2 infections can be induced in pediatric patients. Our important findings provide fundamental knowledge on the immune memory responses to SARS-CoV-2 in recovered pediatric patients.

Accurate Diagnosis of COVID-19 by a Novel Immunogenic Secreted SARS-CoV-2 orf8 Protein.

An accurate diagnostic test for early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the key weapon to control the coronavirus disease 2019 (COVID-19) pandemic. We previously reported that the SARS-CoV-2 genome contains a unique orf8 accessory gene absent from other human-pathogenic coronaviruses. Here, we characterized the SARS-CoV-2 orf8 as a novel immunogenic secreted protein and utilized it for the accurate diagnosis of COVID-19. Extracellular orf8 protein was detected in cell culture supernatant and in sera of COVID-19 patients. In addition, orf8 was found highly immunogenic in COVID-19 patients, who showed early seropositivity for anti-orf8 IgM, IgG, and IgA. We hypothesize that orf8 secretion during SARS-CoV-2 infection facilitates early mounting of B cell response. The serological test detecting anti-orf8 IgG antibody can be used for the early and accurate diagnosis of COVID-19.IMPORTANCE Current commercially available serological tests for COVID-19 patients are detecting antibodies against SARS-CoV-2 nucleoprotein and spike glycoprotein. The antinucleoprotein and antispike antibodies can be accurately detected in patients during the mid or late stage of infection, and therefore, these assays have not been widely used for early diagnosis of COVID-19. In this study, we characterized the secretory property of a SARS-CoV-2 orf8 protein and proposed that orf8 secretion during infection facilitates early mounting of the B cell response. We demonstrated the presence of anti-orf8 antibodies in both symptomatic and asymptomatic patients during the early stage of infection, while the anti-N antibody is not detected. Our serological test detecting anti-orf8 antibodies may facilitate the development of early and accurate diagnosis for COVID-19.

A broad-spectrum virus- and host-targeting peptide against respiratory viruses including influenza virus and SARS-CoV-2.

The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses.

COVID-19 salivary signature: diagnostic and research opportunities.

The COVID-19 (caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) epidemic started in Wuhan (Hubei Province, China) in mid-December 2019 and quickly spread across the world as a pandemic. As a key to tracing the disease and to implement strategies aimed at breaking the chain of disease transmission, extensive testing for SARS-CoV-2 was suggested. Although nasopharyngeal/oropharyngeal swabs are the most commonly used biological samples for SARS-CoV-2 diagnosis, they have a number of limitations related to sample collection and healthcare personnel safety. In this context, saliva is emerging as a promising alternative to nasopharyngeal/oropharyngeal swabs for COVID-19 diagnosis and monitoring. Saliva collection, being a non-invasive approach with possibility for self-collection, circumvents to a great extent the limitations associated with the use of nasopharyngeal/oropharyngeal swabs. In addition, various salivary biomarkers including the salivary metabolomics offer a high promise to be useful for better understanding of COVID-19 and possibly in the identification of patients with various degrees of severity, including asymptomatic carriers. This review summarises the clinical and scientific basis for the potential use of saliva for COVID-19 diagnosis and disease monitoring. Additionally, we discuss saliva-based biomarkers and their potential clinical and research applications related to COVID-19.

Absence of nosocomial transmission of coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 in the prepandemic phase in Hong Kong.

BACKGROUND: To describe the infection control strategy to achieve zero nosocomial transmission of symptomatic coronavirus disease (COVID-19) due to SARS-CoV-2 during the prepandemic phase (the first 72 days after announcement of pneumonia cases in Wuhan) in Hong Kong. METHODS: Administrative support with the aim of zero nosocomial transmission by reducing elective clinical services, decanting wards, mobilizing isolation facilities, providing adequate personal protective equipment, coordinating laboratory network for rapid molecular diagnosis under 4-tier active surveillance for hospitalized patients and outpatients, and organizing staff forum and training was implemented under the framework of preparedness plan in Hospital Authority. The trend of SARS-CoV-2 in the first 72 days was compared with that of SARS-CoV 2003. RESULTS: Up to day 72 of the epidemic, 130 (0.40%) of 32,443 patients being screened confirmed to have SARS-CoV-2 by reverse transcription polymerase chain reaction. Compared with SARS outbreak in 2003, the SARS-CoV-2 case load constituted 8.9% (130 SARS-CoV-2/1458 SARS-CoV) of SARS-CoV infected cases at day 72 of the outbreak. The incidences of nosocomial acquisition of SARS-CoV per 1,000 SARS-patient-day and per 100 SARS-patient-admission were 7.9 and 16.9, respectively, which were significantly higher than the corresponding incidences of SARS-CoV-2 (zero infection, P <.001). CONCLUSIONS: Administrative support to infection control could minimize the risk of nosocomial transmission of SARS-CoV-2.

Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a Hong Kong Cohort: Systematic Review and Meta-analysis.

BACKGROUND & AIMS: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which has been characterized by fever, respiratory, and gastrointestinal symptoms as well as shedding of virus RNA into feces. We performed a systematic review and meta-analysis of published gastrointestinal symptoms and detection of virus in stool and also summarized data from a cohort of patients with COVID-19 in Hong Kong. METHODS: We collected data from the cohort of patients with COVID-19 in Hong Kong (N = 59; diagnosis from February 2 through February 29, 2020),and searched PubMed, Embase, Cochrane, and 3 Chinese databases through March 11, 2020, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We analyzed pooled data on the prevalence of overall and individual gastrointestinal symptoms (loss of appetite, nausea, vomiting, diarrhea, and abdominal pain or discomfort) using a random effects model. RESULTS: Among the 59 patients with COVID-19 in Hong Kong, 15 patients (25.4%) had gastrointestinal symptoms, and 9 patients (15.3%) had stool that tested positive for virus RNA. Stool viral RNA was detected in 38.5% and 8.7% among those with and without diarrhea, respectively (P = .02). The median fecal viral load was 5.1 log10 copies per milliliter in patients with diarrhea vs 3.9 log10 copies per milliliter in patients without diarrhea (P = .06). In a meta-analysis of 60 studies comprising 4243 patients, the pooled prevalence of all gastrointestinal symptoms was 17.6% (95% confidence interval [CI], 12.3-24.5); 11.8% of patients with nonsevere COVID-19 had gastrointestinal symptoms (95% CI, 4.1-29.1), and 17.1% of patients with severe COVID-19 had gastrointestinal symptoms (95% CI, 6.9-36.7). In the meta-analysis, the pooled prevalence of stool samples that were positive for virus RNA was 48.1% (95% CI, 38.3-57.9); of these samples, 70.3% of those collected after loss of virus from respiratory specimens tested positive for the virus (95% CI, 49.6-85.1). CONCLUSIONS: In an analysis of data from the Hong Kong cohort of patients with COVID-19 and a meta-analysis of findings from publications, we found that 17.6% of patients with COVID-19 had gastrointestinal symptoms. Virus RNA was detected in stool samples from 48.1% patients, even in stool collected after respiratory samples had negative test results. Health care workers should therefore exercise caution in collecting fecal samples or performing endoscopic procedures in patients with COVID-19, even during patient recovery.

Development and evaluation of a conventional RT-PCR for differentiating emerging influenza B/Victoria lineage viruses with hemagglutinin amino acid deletion from B/Yamagata lineage viruses.

BACKGROUND: Recent influenza B/Victoria lineage viruses contain amino acid deletions at positions 162 to 164 of the haemagglutinin (HA) protein. These amino acid deletions have affected the detection of B/Victoria lineage viruses by the lineage-specific conventional reverse-transcription polymerase chain reaction (RT-PCR) that was recommended by World Health Organization (WHO). OBJECTIVES: We aimed to develop and evaluate a novel lineage-specific RT-PCR for rapid differentiation of the contemporary B/Victoria lineage from B/Yamagata lineage viruses. STUDY DESIGN: Primers of our in-house RT-PCR were designed to avoid amino acid positions 162 to 164 and to target conserved regions of the HA gene that are specific for B/Victoria and B/Yamagata lineage viruses. Our in-house RT-PCR and WHO RT-PCR were evaluated using influenza B positive clinical specimens or virus culture isolates. Influenza B virus lineage was confirmed by Sanger sequencing. RESULTS: A total of 105 clinical specimens or virus culture isolates were retrieved, including 83 with B/Victoria lineage and 22 with B/Yamagata lineage viruses. Our in-house RT-PCR correctly identified B/Victoria lineage viruses in all 83 samples, including 82 samples with double or triple amino acid deletion in the HA protein. Conversely, the WHO lineage-specific conventional RT-PCR failed to detect any of the 82 samples with HA amino acid deletions. For the 22 samples with B/Yamagata lineage viruses, both RT-PCR assays have correctly identified B/Yamagata lineage in all samples. CONCLUSIONS: Our novel lineage-specific RT-PCR has successfully detected all contemporary B/Victoria lineage viruses with amino acid deletions in HA. This protocol is especially useful for laboratories without the equipment for real-time PCR.

Assessment of population susceptibility to upcoming seasonal influenza epidemic strain using interepidemic emerging influenza virus strains.

Seasonal influenza virus epidemics have a major impact on healthcare systems. Data on population susceptibility to emerging influenza virus strains during the interepidemic period can guide planning for resource allocation of an upcoming influenza season. This study sought to assess the population susceptibility to representative emerging influenza virus strains collected during the interepidemic period. The microneutralisation antibody titers (MN titers) of a human serum panel against representative emerging influenza strains collected during the interepidemic period before the 2018/2019 winter influenza season (H1N1-inter and H3N2-inter) were compared with those against influenza strains representative of previous epidemics (H1N1-pre and H3N2-pre). A multifaceted approach, incorporating both genetic and antigenic data, was used in selecting these representative influenza virus strains for the MN assay. A significantly higher proportion of individuals had a ⩾four-fold reduction in MN titers between H1N1-inter and H1N1-pre than that between H3N2-inter and H3N2-pre (28.5% (127/445) vs. 4.9% (22/445), P < 0.001). The geometric mean titer (GMT) of H1N1-inter was significantly lower than that of H1N1-pre (381 (95% CI 339-428) vs. 713 (95% CI 641-792), P < 0.001), while there was no significant difference in the GMT between H3N2-inter and H3N2-pre. Since A(H1N1) predominated the 2018-2019 winter influenza epidemic, our results corroborated the epidemic subtype.

H7N9 influenza A virus activation of necroptosis in human monocytes links innate and adaptive immune responses.

We previously demonstrated that avian influenza A H7N9 virus preferentially infected CD14+ monocyte in human peripheral blood mononuclear cells (PBMCs), which led to apoptosis. To better understand H7N9 pathogenesis in relation to monocyte cell death, we showed here that extensive phosphorylation of mixed lineage kinase domain-like (MLKL) protein occurred concurrently with the activation of caspases-8, -9 and -3 in H7N9-infected monocytes at 6 h post infection (hpi), indicating that apoptosis and necroptosis pathways were simultaneously activated. The apoptotic morphology was readily observed in H7N9-infected monocytes with transmission electron microscopy (TEM), while the pan-caspase inhibitor, IDN6556 (IDN), accelerated cell death through necroptosis as evidenced by the increased level of pMLKL accompanied with cell swelling and plasma membrane rupture. Most importantly, H7N9-induced cell death could only be stopped by the combined treatment of IDN and necrosulfonamide (NSA), a pMLKL membrane translocation inhibitor, but not by individual inhibition of caspase or RIPK3. Our data further showed that activation of apoptosis and necroptosis pathways in monocytes differentially contributed to the immune response of monocytes upon H7N9 infection. Specifically, caspase inhibition significantly enhanced, while RIPK3 inhibition reduced the early expression of type I interferons and cytokine/chemokines in H7N9-infected monocytes. Moreover, culture supernatants from IDN-treated H7N9-infected monocyte promoted the expression of co-stimulatory molecule CD80, CD83 and CD86 on freshly isolated monocytes and monocyte-derived dendritic cells (MDCs) and enhanced the capacity of MDCs to induce CD3+ T-cell proliferation in vitro. In contrast, these immune stimulatory effects were abrogated by using culture supernatants from H7N9-infected monocyte with RIPK3 inhibition. In conclusion, our findings indicated that H7N9 infection activated both apoptosis and necroptosis in monocytes. An intact RIPK3 activity is required for upregulation of innate immune responses, while caspase activation suppresses the immune response.