1,721,348 research outputs found
Rhinovirus infection in neuronal cells and brain
No full textTraditionally, rhinovirus is considered to cause common cold or mild upper respiratory tract infection. Recently, rhinovirus is frequently reported to cause lower respiratory tract infection and exacerbation of underlying chronic lung diseases. Moreover, there is accumulating evidence that rhinovirus is associated with extrapulmonary manifestations.
A previous study showed that 23% of critically ill patients with rhinovirus infection had seizure. The aim of this study was to systematically investigate rhinovirus infection of the central nervous system (CNS) in vitro and in vivo.
In chapter 4, we assessed the infection of rhinovirus in neuronal cell lines. First, rhinovirus 1A, a minor group rhinovirus, was found to replicate in three different neuronal cell lines (SK-N-SH, NT2 and SF268). In multicycle growth assay by RT-qPCR and plaque assay, rhinovirus 1A replicated best in the neuroblastoma cell line SK-N-SH. Infection in SK-N-SH was confirmed by viral antigen detection with immunofluorescent assay, and cytopathic effect was observed as early as day 1 post-infection. Second, we compared the replication of minor and major group rhinoviruses in SK-N-SH. In the multicycle growth assay of SK-N-SH cells, the viral load on day 3 post-infection was significantly higher for the minor group rhinovirus 1A than those of the major group rhinoviruses A16 and B14.
In chapter 5, the host cytokine response and mode of cell death were assessed. Rhinovirus 1A infection induced the expression of proinflammatory cytokines in neuronal cells, including IL-1β, IL-6 and TNF-α. As shown in the TUNEL assay, rhinovirus 1A infection induced apoptosis in SK-N-SH.
In chapters 6 and 7, a mouse model was used to investigate rhinovirus infection in the central nervous system. Enterovirus D68, a neurotropic virus previously known as rhinovirus 87, was used as a positive control in the mouse experiments. Intracerebral inoculation of rhinovirus 1A resulted in >10% body weight loss. When compared with the neuropathic enterovirus D68, intracerebral inoculation of rhinovirus 1A resulted in a higher viral load and a more prolonged infection, but a lower level of cytokine expression in brain tissues. Mice with intracerebral inoculation of rhinovirus A16 or B14 did not exhibit any weight loss or symptoms. Intranasal inoculation of rhinovirus 1A resulted in pneumonia, but there was no evidence of central nervous system infection after intranasal inoculation of rhinovirus 1A.
In summary, although rhinovirus can infect neuronal cells and induce proinflammatory response in vitro and in vivo, infection via the respiratory route did not result in CNS invasion. Our results corroborate with the clinical findings in which rhinovirus is seldom identified in the cerebrospinal fluid or brain tissue of patients with rhinovirus infection. However, the ability of rhinovirus to infect neuronal cells suggests that rhinovirus may indeed infect CNS in immunocompromised patients.published_or_final_versionMicrobiologyMasterMaster of Philosoph
Miltefosine as a novel influenza vaccine adjuvant
No full textVaccine plays a major role in the prevention of influenza virus infection. However, the effectiveness of currently available influenza vaccines is unsatisfactory. According to the United States Centers for Disease Control and Prevention, the effectiveness of the influenza vaccine fell below 50% for H3N2 in most seasons in the past 15 years. Furthermore, the influenza vaccine generally induces a protective immune response at least two weeks after vaccination. Therefore there is a window period without protection shortly after vaccination.
In this study, miltefosine, an FDA-approved anti-parasitic drug, was evaluated as a novel influenza vaccine adjuvant. I found that the miltefosine-adjuvanted vaccine improved the vaccine-induced protection in mice upon lethal virus challenge at 3 or 7 days after vaccination. The significantly higher survival rate, lower body weight loss, and milder lung damage were observed in infected mice receiving the miltefosine-adjuvanted vaccine (VCM) compared to those receiving vaccine alone (Vac).
To decipher the beneficial effect of miltefosine, the immune responses were studied. The antibody titers were significantly higher in mice receiving VCM than Vac before and after the virus challenge. Besides, post-infection sera from the VCM group were found to be cross-reactive against an antigenically-drifted virus strain, suggesting the induction of cross-protection by VCM. Antigen-specific splenocyte cytokine responses were also significantly increased in mice receiving VCM. The frequencies of TFH cells elevated significantly in mice receiving VCM compared to those receiving Vac, before and after the virus challenge. Histological examination showed that the formation of germinal centers was also promoted by miltefosine-adjuvanted vaccination.
Superior immunological memory was induced by VCM compared to that of Vac. Quicker and greater secondary antibody responses were observed in mice given VCM upon recall of vaccine antigens.
To further elucidate the mechanisms of adjuvant effects of miltefosine, whole blood transcriptomic analysis was conducted. After miltefosine-adjuvanted vaccination, increased oxidative stress responses and erythrocytes and hemoglobin activity were observed. These were associated with improved vaccine effectiveness directly or indirectly through inflammatory responses. After virus challenge, reduced inflammatory responses, increased platelet activation, and upregulated actin cytoskeleton activity were found in the VCM group. These were linked to reduced lung tissue damage and promoted activation of monocytes and adaptive immunity.
The vaccine formula based on a cocktail of three adjuvants with influenza vaccine, including miltefosine, imiquimod, and paromomycin, were found to increase the vaccine effectiveness, but the effect was not better than miltefosine adjuvanted influenza vaccine. Additionally, miltefosine was demonstrated not to be an antiviral drug against the influenza virus.
Collectively, the miltefosine-adjuvanted influenza vaccine showed significantly improved protection in mice with only one dose and in a much shorter time. The improved protective response is related to augmented antibody response and TFH cell-associated germinal center response. Comparing to other vaccine adjuvants, miltefosine has the advantage of being a clinically-approved drug with known safety data in human. Repurposing existing drugs is a feasible option for identifying novel vaccine adjuvants.published_or_final_versionMicrobiologyDoctoralDoctor of Philosoph
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Antibody response in influenza and COVID-19
No full textSince 1918, influenza virus has led to four pandemics. In 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the COVID-19 pandemic. Both influenza virus and SARS-CoV-2 continue to cause surges in hospitalizations after the pandemic. The studies in this thesis sought to determine population immunity against these viruses.
The risk of influenza was determined by assessing the neutralizing antibody (NAb) titers against the circulating strains just before each influenza season. In 2018 summer, the geometric mean NAb titers (GMT) against a circulating A(H1N1) strain was lower than a strain collected during the prior epidemic, which predicted the dominance of A(H1N1) in the 2018/2019 winter influenza season. In April-June 2019, the GMT against the circulating A(H1N1), A(H3N2) and influenza B viruses significantly decreased, which coincided with the co-circulation of the three influenza subtypes in 2019 summer. Hence, seroprevalence data are helpful in forecasting the dominant strains of the upcoming influenza season.
SARS-CoV-2 emerged during the 2019/2020 winter. The newly developed enzyme immunoassay (EIA) and conventional virus neutralization test (cVNT) were used for assessing the antibody response of COVID-19 patients. During the acute phase, immunoglobulin M (IgM) and immunoglobulin G (IgG) against nucleoprotein (N) and spike protein receptor binding domain (RBD) could be detected in most individuals at 90% of patients at 12 months PSO.
The seroprevalence of COVID-19 in Hong Kong before and during the COVID-19 pandemic, and among Hong Kong residents evacuated from Hubei province in March 2020, were determined. Among the 1938 sera collected in Hong Kong before March 2020, none tested positive for both EIA and cVNT, indicating that SARS-CoV-2 did not circulate in Hong Kong. Among the 452 returnees from Hubei province, 3.3% tested positive with both EIA and cVNT which was much higher than the prevalence of RT-PCR confirmed cases, suggesting that the majority of COVID-19 patients had subclincial infection.
As SARS-CoV-2 variants emerged, the risk of these variants to the Hong Kong population was assessed by testing the serum antibody titers of vaccinated or infected individuals. The NAb titers against the Beta, Theta, and Kappa were 3-7 fold lower than those against the ancestral strain. Our mechanistic study showed that RBD with mutation at the amino acid residue 484 affected the antibody binding, indicating that mutation at this site was critical for immune escape.
The Omicron variant, which led to the devastating fifth wave in Hong Kong in early 2022, was most immunoevasive, with a >20-fold reduction in serum NAb titers. Our seroprevalence data showed that only <10% of the Hong Kong population had detectable NAb against the Omicron variant before the fifth wave. In particular, none of the older adults aged ≥70 years old had detectable levels of NAb against the Omicron variant.
The studies in this thesis illustrated the importance of serological surveillance in determining population immunity. Continual serological surveillance would be crucial in risk assessment, guiding public health measures.published_or_final_versionMicrobiologyDoctoralDoctor of Philosoph
The molecular evolution of SARS-CoV-2
No full textThe COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed unprecedented challenges to global public health, economies, and societies. This thesis aims to understand the transmission dynamics, evolution, and antiviral resistance of SARS-CoV-2 through next-generation sequencing.
In Chapter 3, viral genomic studies were used to investigate 3 outbreaks in Hong Kong. Using whole genome sequencing and phylogenetic analysis, the sources of the outbreak were identified, including 1) an imported case from Nepal that caused wave 4 in November 2020; 2) imported cases from travelers returning to Hong Kong that caused a housing estate outbreak due to Omicron variant in January 2022; and 3) imported hamsters from the Netherlands that cause an outbreak f Delta variant AY.127. These findings revealed the rapid spread of B.1.36.27 and Omicron variants in Hong Kong, the effectiveness of timely nonpharmaceutical interventions (NPIs) in curbing transmission, and the limitation of stringent public health measures. This Chapter also explored the transmission of SARS-CoV-2 from pet hamsters to humans, providing evidence of zoonotic transfer, and its implications for public health.
In Chapter 4, the emergence of amino acid substitutions during acute or chronic SARS-CoV-2 infection was accessed. In Chapter 4.1, in a patient with acute SARS-CoV-2 infection, the emergence of amino acid substitution in a neutralizing epitope at the N-terminal of Spike protein was found. In Chapter 4.2, de novo amino acid substitutions (DNS) in 34 chronically infected patients was identified, including 4 patients from our hospital. Many DNS that appeared in multiple patients were lineage-defining mutations of epidemiologically important SARS-CoV-2 variants, such as the Omicron variant. Most importantly, among the 4 chronically infected patients in our hospital, nsp9 T35I was found to have emerged in 3 of them. The mutation may affect the functional interaction with nsp12 in the replication and transcription complex.
In Chapter 5, the prevalence of antiviral resistance was evaluated using sequence data deposited in a public database (GISAID) up to December 2022, 1 year after the approval of Paxlovid, a 3CL protease inhibitor. We found that 0.5% (67,095/13,446,588) of the SARS-CoV-2 sequences contain nsp5 substitutions that were known to confer resistance against the 3CL protease (nsp5) inhibitors. Further analysis did not identify any particular nsp5 substitutions that are increasing in prevalence globally.
In conclusion, this thesis contributes to the understanding of the molecular evolution of SARS-CoV-2 by applying genomics sequencing to the epidemiological investigation of outbreaks, offering valuable insights into its transmission dynamics, the impact of control measures, intrahost variation, and the challenges posed by zoonotic transfer. The findings also emphasized the critical role of genomic surveillance in detecting antiviral drug resistance in a population.published_or_final_versionMicrobiologyDoctoralDoctor of Philosoph
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Clinical application of target amplicon sequencing for rapid diagnosis of drug resistant mycobacterium tuberculosis in respiratory specimens
No full textTuberculosis (TB) has been a global public health concern for more than a century. It remains one of the top 10 causes of death worldwide and the leading cause of death from a single infectious agent. Situation is worsened due to the emerging of multi-drug resistant TB (MDR-TB) and even extensively drug resistant TB (XDR-TB), dwarfing the effectiveness of current anti-TB treatment regime and the continuous effort towards TB infection control globally. Advancement in molecular diagnostics could provide a lot more rapid and accurate TB diagnosis and also drug-resistance detection comparing to the conventional methods including culture and phenotypic drug susceptibility test (pDST). This study aimed to evaluate a protocol for rapid TB diagnosis and drug resistance detection, with preliminary screening of MTB positive cases with IS6110 real-time PCR, followed by TB-drug resistance detection target amplicon sequencing NGS.
A total of 337 direct respiratory samples were collected during August 2020 to March 2021. All samples were tested with IS6110 real-time PCR and 76 samples tested with Ct values ranging 4.93 to 28.44 were selected for target amplicon sequencing, which covers 18 gene targets conferring anti-TB drug resistance. Target amplicon sequencing was performed using Illumina MiSeq on sample DNA purified and concentrated with our in-house validated protocol. Among the 76 selected samples, 69 of which were successfully sequenced and passed our defined acceptance criteria of 500X sequencing depth. Drug-resistance associated mutation(s) were detected in 17 samples with mutation frequency >95% and 1 sample with low mutation frequency at 37.04%, while no mutation was detected for the remaining 53 samples. Among the 17 samples detected with drug-resistance associated mutation(s) at frequency >95%, 10 of which were mono-drug resistant TB while the other 6 were multi-drug resistant TB. There were also 3 multi-drug resistant TB samples carrying mutations associated to fluoroquinolones (FQs) and considered as pre-XDR-TB. Concordant result was obtained in 16/17 (94.1%) when comparing the major variants at mutation frequency >95% with pDST results. One phenotypic pan-susceptible case was detected to harbour rpoB L533P mutation. The low variant frequency detected was phenotypic pan-susceptible. It could be well distinguished from the phenotypic resistant cases by variant frequency and hence the minor variants detected would not affect the overall result interpretation for target amplicon sequencing towards TB drug-resistance detection.
This evaluated protocol, with preliminary screening for IS6110 positive cases, followed by subsequent target amplicon sequencing for detection of drug-resistance associated mutations, could provide a rapid, accurate and cost-effective diagnostic solution for drug-resistance TB cases with turn-around time of around 10 working days. Which shortened the turn-around time a lot when comparing to the conventional methods including culture and phenotypic drug susceptibility test (pDST) which require 8 to 12 weeks. This protocol is also user friendly and easy to be adopted as routine testing by clinical laboratory worldwide. The timely genotypic resistance profile obtained from the target amplicon sequencing could facilitate clinician to have better and timely treatment planning for drug resistant TB patients and improve the epidemiology control of drug resistant TB.published_or_final_versionMicrobiologyMasterMaster of Philosoph
Molecular epidemiology of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae
No full textIncreasing carbapenem resistance among clinical isolates of E. coli and K. pneumoniae has become a serious public health problem over the last decade. Molecular epidemiology studies have shown that there is a global dissemination of epidemic clones of carbapenem-resistant E. coli and K. pneumoniae. Besides, successful epidemic plasmids were reported to disseminate carbapenemase genes in Enterobacteriaceae. The wide spread of carbapenem-resistant E. coli and K. pneumoniae limits treatment options of the infection, poses severe challenges to clinical professionals and threatens our health.
Recently, carbapenem-resistant E. coli and K. pneumoniae are increasingly reported in Hong Kong. In 2012, our group has documented the emergence of carbapenem-resistant clinical isolates in Hong Kong. The findings of the previous study showed that 26.1% of the Enterobacteriaceae isolates were confirmed to produce carbapenemase. Notably, a novel IncX3 plasmid was found to be involved in the dissemination of blaNDM-1 gene. However, the previous findings fail to explicate the carbapenem resistance mechanisms of the remaining non-carbapenemase producing isolates. Further investigation is needed to elucidate the situation.
Firstly, we investigated the carbapenem resistance mechanism of carbapenem-resistant E. coli and K. pneumoniae isolates recovered from the Hong Kong West Cluster hospitals from 2010 to 2012. PCRs were used to detect carbapenemase genes (blaNDM, blaKPC, blaIMP, blaVIM and blaOXA-48), blaCTX-M ESBL genes and blaAmpC genes. SDS-PAGE was used to detect porin loss. Among the 92 isolates in this study, only nine (9.8 %) isolates were detected with carbapenemase genes. The blaCTX-M and/or blaAmpC β-lactamase genes plus porin loss were detected in 47 non-carbapenemase-producing isolates (16 E. coli and 31 K. pneumoniae). The resistance determinant profiles of these 16 E. coli included: blaCTX-M + porin loss (n= 10), blaCIT + porin loss (n = 1), blaCTX-M + blaCIT/DHA + porin loss (n = 5). The resistance determinant profiles of the 31 K. pneumoniae included: blaCTX-M + porin loss (n= 4), blaDHA + porin loss (n = 7), blaCTX-M + blaCIT/DHA + porin loss (n = 20). The results showed that apart from acquired carbapenemases, the production of AmpC β-lactamase and/or ESBLs plus porin loss played a main role in the carbapenem resistance mechanism of the carbapenem-resistant E. coli and K. pneumoniae isolates.
Secondly, we accessed the clonal relatedness of the isolates. Multi-locus sequence typing results showed that 55 (77.5%) K. pneumoniae isolates fall into the clonal complex 37. Our results suggest that the CC37 K. pneumoniae are associated with the acquisition of DHA-1 β-lactamase, CTXM-1-group β-lactamase and porin alterations which could confer a high-level of resistance to carbapenems resulting in their predominance in this study.
Finally, we characterized the plasmids that carry carbapenemase gene by S1-PFGE, Southern blot, plasmid replicon typing and whole plasmid sequencing. A novel IncX3 plasmid was found to carry blaKPC gene. Together with the previously reported blaNDM-1 carrying IncX3 plasmids, it shows that IncX3 plasmids might be new epidemic plasmids involved in the dissemination of carbapenemase genes. These novel IncX3 plasmids are worrisome. Nationwide surveillance and more epidemiological study of IncX3 plasmids are needed.
(Wordpublished_or_final_versionMicrobiologyMasterMaster of Philosoph
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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