Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq.

Klebsiella pneumoniae is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent K. pneumoniae. The discovery and study of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern due to their restriction in the development of novel antibiotics. However, the lack of essential functional genomic data has hampered the study of the mechanisms of essential genes related to antimicrobial susceptibility. In this study, we developed a pooled CE genes mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference screening method (Mobile-CRISPRi-seq) for K. pneumoniae to identify genes that play critical roles in antimicrobial fitness in vitro and host immunity in vivo. Targeting 870 predicted CE genes in K. pneumoniae, Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes folB and folP under trimethoprim pressure. Our screening also identified genes waaE and fldA related to polymyxin and ß-lactam susceptibility by applying a screening strategy based on Mobile-CRISPRi-seq and comparative genomics. Furthermore, using a mouse infection model and Mobile-CRISPRi-seq, multiple virulence genes were identified, and among these genes, pal, yciS, and ribB were demonstrated to contribute to the pathogenesis of K. pneumoniae. This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in K. pneumoniae, and this broadly applicable system can be expanded for high-throughput functional gene study in multiple pathogenic bacteria, especially in gram-negative bacteria. IMPORTANCE The discovery and investigation of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern because of the restriction of antimicrobial targets in the development of novel antibiotics. In this study, we developed a pooled CE gene-wide mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference sequencing (Mobile-CRISPRi-seq) strategy in Klebsiella pneumoniae to identify genes that play critical roles in the fitness of antimicrobials in vitro and host immunity in vivo. The data suggest a robust tool to screen for loss-of-function phenotypes in a pooled gene knockdown library in K. pneumoniae, and Mobile-CRISPRi-seq may be expanded to multiple bacteria for screening and identification of genes with crucial roles in the fitness of antimicrobials and hosts.

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus.

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.

Serosurvey in SARS-CoV-2 inactivated vaccine-elicited neutralizing antibodies against authentic SARS-CoV-2 and its viral variants.

Various variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been emerging and circulating in different parts of the world. Millions of vaccine doses have been administered globally, which reduces the morbidity and mortality of coronavirus disease-2019 efficiently. Here, we assess the immune responses of individuals after two shots of BBIBP-CorV or CoronaVac inactivated vaccine. We measured neutralizing antibody responses after the second vaccination by using authentic SARS-CoV-2 and its viral variants. All the serum samples efficiently neutralized SARS-CoV-2 wild-type lineage, in contrast, a part of serum samples failed to neutralize Alpha, Beta, Gamma, Delta, or Eta lineages, and only several serum samples were able to neutralize Omicron lineage virus strains (BA.1 and BA.2) with low neutralization titer. As compared with the neutralization of SARS-CoV-2 wild-type lineage, the neutralization of all other SARS-CoV-2 variant lineages was significantly lower. Considering that all the SARS-CoV-2 mutation viruses challenged the antibody neutralization induced by BBIBP-CorV and CoronaVac, it is necessary to carry out a third booster vaccination to increase the humoral immune response against the SARS-CoV-2 mutation viruses.

Humoral immune response to authentic circulating severe acute respiratory syndrome coronavirus 2 variants elicited by booster vaccination with distinct receptor-binding domain subunits in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants could induce immune escape by mutations of the spike protein which are threatening to weaken vaccine efficacy. A booster vaccination is expected to increase the humoral immune response against SARS-CoV-2 variants in the population. We showed that immunization with two doses of wild type receptor-binding domain (RBD) protein, and booster vaccination with wild type or variant RBD protein all significantly increased binding and neutralizing antibody titers against wild type SARS-CoV-2 and its variants in mice. Only the booster immunization by Omicron (BA.1)RBD induced a strong antibody titer against the omicron virus strain and comparable antibody titers against all the other virus strains. These findings might shed the light on coronavirus disease 2019 booster immunogens.

Murine diabetic models for dengue virus infection.

Dengue virus (DENV) is a critical public health concern in tropical and subtropical regions worldwide. Thus, immunocompetent murine models of DENV infection with robust viremia are required for vaccine studies. Diabetes is highly prevalent worldwide, making it frequent comorbidity in patients with dengue fever. Therefore, murine models are needed to understand viral pathogenesis and disease progression. Acquired-induced and inherently diabetic C57BL/6 and db/db mice were inoculated with DENV-3 via the tail vein. After infection, both the diabetic C57BL/6 and db/db mice showed obvious weight loss with clinical manifestations. Quantitative reverse-transcription polymerase chain reaction revealed robust and replicable viremia in the two types of diabetic mice. Immunohistochemical detection showed persistent DENV-3 infection in the liver. Enzyme-linked immunosorbent assay for cytokine detection revealed that diabetic mice showed more severe inflammatory responses than did nondiabetic mice, and significant histological alterations were observed in diabetic mice. Thus, the diabetic mice were more susceptible to DENV infection than the nondiabetic mice. Taken together, we established two types of immunocompetent diabetic mice for DENV infection, which can be used to further study the mechanisms of dengue pathogenesis in diabetes and to develop antiviral pharmaceuticals and treatments.

Assessment of antibody responses against SARS-CoV-2 in unvaccinated individuals and vaccinees from Omicron-BA.2 infection in Zhaoqing, Guangdong Province, China.

Currently, the majority of the global population has been vaccinated with the COVID-19 vaccine, and characterization studies of antibodies in vivo from Omicron breakthrough infection and naive infection populations are urgently needed to provide pivotal clues about accurate diagnosis, treatment, and next-generation vaccine design against SARS-CoV-2 infection. We showed that after infection with Omicron-BA.2, the antibody levels of specific IgM against the Wuhan strain and specific IgG against Omicron were not significantly elevated within 27 days of onset. Interestingly, in this study, the levels of humoral immunity against Omicron-specific IgM were significantly increased after breakthrough infection, suggesting that the detection of Omicron-specific IgM antibodies can be used as a test criterion of Omicron breakthrough infection. In addition, we observed that serums from unvaccinated individuals and the majority of vaccinated infections possessed only low or no neutralizing activity against Omicron at the onset of Omicron breakthrough infections, and at the later stage of Omicron-BA.2 breakthrough infection, levels of neutralization antibody against the Wuhan and Omicron strains were elevated in infected individuals. The findings of this study provide important clues for the diagnosis of Omicron breakthrough infections, antibody characterization studies and vaccine design against COVID-19.

Pulmonary fibrosis and its related factors in discharged patients with new corona virus pneumonia: a cohort study.

BACKGROUND: Thousands of Coronavirus Disease 2019 (COVID-19) patients have been discharged from hospitals Persistent follow-up studies are required to evaluate the prevalence of post-COVID-19 fibrosis. METHODS: This study involves 462 laboratory-confirmed patients with COVID-19 who were admitted to Shenzhen Third People's Hospital from January 11, 2020 to April 26, 2020. A total of 457 patients underwent thin-section chest CT scans during the hospitalization or after discharge to identify the pulmonary lesion. A total of 287 patients were followed up from 90 to 150 days after the onset of the disease, and lung function tests were conducted about three months after the onset. The risk factors affecting the persistence of pulmonary fibrosis were identified through regression analysis and the prediction model of the persistence of pulmonary fibrosis was established. RESULTS: Parenchymal bands, irregular interfaces, reticulation and traction bronchiectasis were the most common CT features in all COVID-19 patients. During the 0-30, 31-60, 61-90, 91-120 and > 120 days after onset, 86.87%, 74.40%, 79.56%, 68.12% and 62.03% patients developed with pulmonary fibrosis and 4.53%, 19.61%, 18.02%, 38.30% and 48.98% patients reversed pulmonary fibrosis, respectively. It was observed that Age, BMI, Fever, and Highest PCT were predictive factors for sustaining fibrosis even after 90 days from onset. A predictive model of the persistence with pulmonary fibrosis was developed based-on the Logistic Regression method with an accuracy, PPV, NPV, Sensitivity and Specificity of the model of 76%, 71%, 79%, 67%, and 82%, respectively. More than half of the COVID-19 patients revealed abnormal conditions in lung function after 90 days from onset, and the ratio of abnormal lung function did not differ on a statistically significant level between the fibrotic and non-fibrotic groups. CONCLUSIONS: Persistent pulmonary fibrosis was more likely to develop in patients with older age, higher BMI, severe/critical condition, fever, a longer viral clearance time, pre-existing disease and delayed hospitalization. Fibrosis developed in COVID-19 patients could be reversed in about a third of the patients after 120 days from onset. The pulmonary function of less than half of COVID-19 patients could turn to normal condition after three months from onset. An effective prediction model with an average area under the curve (AUC) of 0.84 was established to predict the persistence of pulmonary fibrosis in COVID-19 patients for early diagnosis.

Intestinal microbiota has important effect on severity of hand foot and mouth disease in children.

BACKGROUND: The incidence of hand foot and mouth disease (HFMD) has increased in recent years, making it a very common childhood illness worldwide. The relationship between different enterovirus genotypes and disease severity is not clearly understood. Given that enteroviruses are transmitted through the gastrointestinal tract, we hypothesized that variation in intestinal microorganisms of the host might play a role in the prognosis of HFMD. METHODS: We carried out a meta-transcriptomic-wide association study of fecal samples obtained from a cohort of children (254 patients, 227 tested positive for enterovirus, including 16 patients co-infectied with 2 kinds of enterovirus) with mild and severe HFMD and healthy controls. RESULTS: We found there was no significant difference in the amount of each virus type between the mild and severe cases. Genes of enterovirus 71 (EV71) and coxsackievirus A (CV-A) from the severe and mild cases did not show significant clustering. Clostridium sp. L2-50 and Bacteroides stercoris ATCC 43183 were enriched in the guts of children with severe HFMD and KEGG enrichment was found between mild and severe cases. CONCLUSIONS: Intestinal microorganisms appear to interact with enterovirus to determine the progression of HFMD. Genes of Bacteroides and Clostridium may be used as predictive markers for a more efficient prognosis and intervention. The enrichment of intestinal bacteria genes with functions may facilitate the development of severe symptoms for HFMD patients.

Plasma IP-10 and MCP-3 levels are highly associated with disease severity and predict the progression of COVID-19.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 was first reported in Wuhan, December 2019, and continuously poses a serious threat to public health, highlighting the urgent need of identifying biomarkers for disease severity and progression. OBJECTIVE: We sought to identify biomarkers for disease severity and progression of COVID-19. METHODS: Forty-eight cytokines in the plasma samples from 50 COVID-19 cases including 11 critically ill, 25 severe, and 14 moderate patients were measured and analyzed in combination with clinical data. RESULTS: Levels of 14 cytokines were found to be significantly elevated in COVID-19 cases and showed different expression profiles in patients with different disease severity. Moreover, expression levels of IFN-γ-induced protein 10, monocyte chemotactic protein-3, hepatocyte growth factor, monokine-induced gamma IFN, and macrophage inflammatory protein 1 alpha, which were shown to be highly associated with disease severity during disease progression, were remarkably higher in critically ill patients, followed by severe and then the moderate patients. Serial detection of the 5 cytokines in 16 cases showed that continuously high levels were associated with deteriorated progression of disease and fatal outcome. Furthermore, IFN-γ-induced protein 10 and monocyte chemotactic protein-3 were excellent predictors for the progression of COVID-19, and the combination of the 2 cytokines showed the biggest area under the curve of the receiver-operating characteristics calculations with a value of 0.99. CONCLUSIONS: In this study, we report biomarkers that are highly associated with disease severity and progression of COVID-19. These findings add to our understanding of the immunopathologic mechanisms of severe acute respiratory syndrome coronavirus 2 infection, and provide potential therapeutic targets and strategies.

Correction to: Intra-host diversity of hepatitis B virus during mother-to-child transmission: the X gene may play a key role in virus survival in children after transmission.

Authors would like to correct few errors in their publication.

Intra-host diversity of hepatitis B virus during mother-to-child transmission: the X gene may play a key role in virus survival in children after transmission.

Mother-to-child transmission of hepatitis B virus (HBV) is the main route of transmission in Asia, and characterization of HBV quasispecies is needed to further understand virus evolution and adaptation. To understand changes in HBV during mother-to-child transmission, we enrolled nine pairs of mothers and children in the study, including a set of twins. Three groups were infected with HBV genotype C, and six groups were infected with HBV genotype B. The full-length HBV genome was amplified by PCR from serum samples before antiviral treatment, the whole viral genomes from each pair were sequenced, and the complexity and diversity of the quasispecies were analyzed. The entropy of transmitted HBV in children was found to be lower than their mothers, suggesting that there was a bottleneck effect during HBV transmission from the mother to the child. Selective evolution was shown by calculating πN and πS in the whole genomes, and the highest values were obtained for the X gene, which plays a role in viral replication and immune escape. All genotype C patients and only one genotype B pair had a πN/πS greater than 1 ratio, indicating that positive selection had occurred. In addition, quasispecies were found to be different between the twin children despite having the same mother, indicating that virus evolution is host-specific.

Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma.

Importance: Coronavirus disease 2019 (COVID-19) is a pandemic with no specific therapeutic agents and substantial mortality. It is critical to find new treatments. Objective: To determine whether convalescent plasma transfusion may be beneficial in the treatment of critically ill patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Design, Setting, and Participants: Case series of 5 critically ill patients with laboratory-confirmed COVID-19 and acute respiratory distress syndrome (ARDS) who met the following criteria: severe pneumonia with rapid progression and continuously high viral load despite antiviral treatment; Pao2/Fio2 <300; and mechanical ventilation. All 5 were treated with convalescent plasma transfusion. The study was conducted at the infectious disease department, Shenzhen Third People's Hospital in Shenzhen, China, from January 20, 2020, to March 25, 2020; final date of follow-up was March 25, 2020. Clinical outcomes were compared before and after convalescent plasma transfusion. Exposures: Patients received transfusion with convalescent plasma with a SARS-CoV-2-specific antibody (IgG) binding titer greater than 1:1000 (end point dilution titer, by enzyme-linked immunosorbent assay [ELISA]) and a neutralization titer greater than 40 (end point dilution titer) that had been obtained from 5 patients who recovered from COVID-19. Convalescent plasma was administered between 10 and 22 days after admission. Main Outcomes and Measures: Changes of body temperature, Sequential Organ Failure Assessment (SOFA) score (range 0-24, with higher scores indicating more severe illness), Pao2/Fio2, viral load, serum antibody titer, routine blood biochemical index, ARDS, and ventilatory and extracorporeal membrane oxygenation (ECMO) supports before and after convalescent plasma transfusion. Results: All 5 patients (age range, 36-65 years; 2 women) were receiving mechanical ventilation at the time of treatment and all had received antiviral agents and methylprednisolone. Following plasma transfusion, body temperature normalized within 3 days in 4 of 5 patients, the SOFA score decreased, and Pao2/Fio2 increased within 12 days (range, 172-276 before and 284-366 after). Viral loads also decreased and became negative within 12 days after the transfusion, and SARS-CoV-2-specific ELISA and neutralizing antibody titers increased following the transfusion (range, 40-60 before and 80-320 on day 7). ARDS resolved in 4 patients at 12 days after transfusion, and 3 patients were weaned from mechanical ventilation within 2 weeks of treatment. Of the 5 patients, 3 have been discharged from the hospital (length of stay: 53, 51, and 55 days), and 2 are in stable condition at 37 days after transfusion. Conclusions and Relevance: In this preliminary uncontrolled case series of 5 critically ill patients with COVID-19 and ARDS, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status. The limited sample size and study design preclude a definitive statement about the potential effectiveness of this treatment, and these observations require evaluation in clinical trials.

Epidemiological trends and virological traits of hepatitis B virus infection in pregnant women and neonates.

In infants, hepatitis B virus (HBV) infections are mainly acquired by mother-to-child transmission (MTCT). Current tests for the presence of HBV markers at birth can neither confirm nor exclude MTCT. The aim of this study was to find an early diagnostic marker of HBV MTCT. From 2011 to 2016, we studied a total of 5999 pregnant women who gave birth at our hospital in Shenzhen City, China. HBsAg-positive mothers and their offspring (n=386 pairs) were tested at birth for HBV markers, and 207 infants were followed up at 7-12 months after birth. The HBsAg-seropositive rate of the pregnant women was 12.5%. Additionally, 28.0%, 36.0%, 98.5% and 6.6% of umbilical cord (UC) blood samples of neonates were found to be positive for HBsAg, HBeAg, anti-HBc and HBV-DNA, respectively, whereas for neonatal femoral venous (FV) blood, the percentages were 16.2%, 38.0%, 98.8% and 2.6%, respectively. Mothers with high HBV DNA loads and those who were HBeAg positive were the most likely to have HBV-positive offspring. Immunoprophylaxis failed in five infants: the difference in median HBV DNA titer between UC blood from infants with and without HBV MTCT was statistically significant, and there was no significant difference in HBV DNA titer between UC blood and in peripheral blood of infants with HBV MTCT. In conclusion, we found that HBeAg positivity and high HBV loads are strong risk factors for MTCT of HBV and that the HBV DNA titer in the UC is a good predictor for HBV MTCT.

Virus-mimetic nanovesicles as a versatile antigen-delivery system.

It is a critically important challenge to rapidly design effective vaccines to reduce the morbidity and mortality of unexpected pandemics. Inspired from the way that most enveloped viruses hijack a host cell membrane and subsequently release by a budding process that requires cell membrane scission, we genetically engineered viral antigen to harbor into cell membrane, then form uniform spherical virus-mimetic nanovesicles (VMVs) that resemble natural virus in size, shape, and specific immunogenicity with the help of surfactants. Incubation of major cell membrane vesicles with surfactants generates a large amount of nano-sized uniform VMVs displaying the native conformational epitopes. With the diverse display of epitopes and viral envelope glycoproteins that can be functionally anchored onto VMVs, we demonstrate VMVs to be straightforward, robust and tunable nanobiotechnology platforms for fabricating antigen delivery systems against a wide range of enveloped viruses.

Enhancing Sensitivity in SARS-CoV-2 Rapid Antigen Testing through Integration of a Water-Soluble Polymer Wall.

Lateral flow immunoassays (LFIAs) are recognized for their practicality in homecare and point-of-care testing, owing to their simplicity, cost-efficiency, and rapid visual readouts. Despite these advantages, LFIAs typically fall short in sensitivity, particularly in detecting viruses such as SARS-CoV-2, thus limiting their broader application. In response to this challenge, we have innovated an approach to substantially enhance LFIA sensitivity. This involves the integration of a water-soluble dextran-methacrylate polymer wall with a 15% grafting degree positioned between the test and control lines on the LFIA strip. This novel modification significantly improved the sensitivity of the assay, achieving detection limits as low as 50 pg mL-1 and enhancing the sensitivity by 5-20-fold relative to existing LFIA kits available on the market. Furthermore, our developed LFIA kit (WSPW-LFIA) demonstrated exceptional specificity for SARS-CoV-2. Coupled with a straightforward fabrication process and robust stability, the WSPW-LFIA represents a promising advancement for real-time in vitro diagnosis across a spectrum of diseases.

SnS Quantum Dots Enhancing Carbon-Based Hole Transport Layer-Free Visible Photodetectors.

The recombination of charges and thermal excitation of carriers at the interface between methylammonium lead iodide perovskite (PVK) and the carbon electrode are crucial factors that affect the optoelectronic performance of carbon-based hole transport layer (HTL)-free perovskite photodetectors. In this work, a method was employed to introduce SnS quantum dots (QDs) on the back surface of perovskite, which passivated the defect states on the back surface of perovskite and addressed the energy-level mismatch issue between perovskite and carbon electrode. Performance testing of the QDs and the photodetector revealed that SnS QDs possess energy-level structures that are well matched with perovskite and have high absorption coefficients. The incorporation of these QDs into the interface layer effectively suppresses the dark current of the photodetector and greatly enhances the utilization of incident light. The experimental results demonstrate that the introduction of SnS QDs reduces the dark current by an order of magnitude compared to the pristine device at 0 V bias and increases the responsivity by 10%. The optimized photodetector exhibits a wide spectral response range (350 nm to 750 nm), high responsivity (0.32 A/W at 500 nm), and high specific detectivity (>1 × 1012 Jones).

Analysis of binding and authentic virus-neutralizing activities of immune sera induced by various monkeypox virus antigens.

Monkeypox cases continue to increase globally, and there is an urgent need to develop a highly effective vaccine against monkeypox. This study investigated the binding and authentic-virus neutralizing activities of sera from mice immunized with EEV (extracellularly enveloped viruses) antigens B6R and A35R, and IMV (intrinsic material viruses) antigens M1R, A29L, E8L, and H3L against monkeypox virus. The results showed that immunizations of A35R and E8L could only induce lower titers of binding antibodies, in contrast, immunization of M1R induced the highest titers of binding antibodies, while immunization of B6R, H3L, and A29L induced moderate titers of binding antibodies. For the live monkeypox virus neutralization assay, the results showed that immunization with two doses of EEV antigen B6R did not effectively induce humoral immune responses to neutralize monkeypox live virus, immunization with EEV-A35R only induced weak monkeypox-neutralizing antibodies. In contrast, the immunization of the four types of monkeypox virus IMV antigens can all induce neutralizing antibodies against authentic monkeypox virus, among them, A29L and H3L induced the highest neutralizing antibody titers. The results of this study provide important references for the selection of antigens in the development of the next generation of monkeypox vaccines.

Development of a quadruple qRT-PCR assay for simultaneous identification of hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

IMPORTANCE: Globally, the increasing number of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp.