published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

jdelvall@upc.edu.pe / Acute respiratory infections are responsible for high morbi–mortality in Peruvian children. However, the etiological agents are poorly identified. This study, conducted during the pandemic outbreak of H1N1 influenza in 2009, aims to determine the main etiological agents responsible for acute respiratory infections in children from Lima, Peru. Nasopharyngeal swabs collected from 717 children with acute respiratory infections between January 2009 and December 2010 were analyzed by multiplex RT-PCR for 13 respiratory viruses: influenza A, B, and C virus; parainfluenza virus (PIV) 1, 2, 3, and 4; and human respiratory syncytial virus (RSV) A and B, among others. Samples were also tested with direct fluorescent-antibodies (DFA) for six respiratory viruses. RT-PCR and DFA detected respiratory viruses in 240 (33.5%) and 85 (11.9%) cases, respectively. The most common etiological agents were RSV-A (15.3%), followed by influenza A (4.6%), PIV-1 (3.6%), and PIV-2 (1.8%). The viruses identified by DFA corresponded to RSV (5.9%) and influenza A (1.8%). Therefore, respiratory syncytial viruses (RSV) were found to be the most common etiology of acute respiratory infections. The authors suggest that active surveillance be conducted to identify the causative agents and improve clinical management, especially in the context of possible circulation of pandemic viruses

O vírus respiratório sincicial humano (HRSV) é principal agente causador de infecções do trato respiratório inferior em crianças e lactentes. Um diagnóstico rápido e preciso evitaria o uso desnecessário de antibióticos, nos casos em que a infecção é viral. A reação em cadeia da polimerase após transcrição reversa (RT-PCR) e o ensaio de imunofluorescência indireta (IFI) são considerados ferramentas importantes na detecção do HRSV, pela alta sensibilidade e especificidade. Visando simplificar e minimizar os riscos de contaminação freqüentes, em duas etapas, foi padronizada uma reação em tubo único para detecção do HRSV em amostras clínicas. Aspirados de nasofaringe de 226 crianças de 0-5 anos de idade, com doença respiratória, atendidas no Hospital Universitário da Universidade de São Paulo (HU-USP), foram testados por imunofluorescência indireta, RT semi Nested PCR e RT-PCR em tubo único. Cento e duas amostras (45,1%) foram positivas em pelo menos uma das técnicas e 75 (33,2%) em todas. Três (1,3%) amostras foram positivas por IFI e RT semi Nested PCR, 1 (0,4%) foi positiva por IFI e RT-PCR em tubo único, 5 (2,2%) amostras foram positivas somente por IFI, 2 (0,9%) somente por RT semi Nested PCR e 16 (7,1%) amostras foram positivas pela RT semi Nested PCR e RT-PCR em tubo único. A RT-PCR em tubo único mostrou ser uma técnica rápida, sensível e específica, e o uso combinado de dois métodos aumenta a detecção do HRSV. / Respiratory Syncytial Virus is the main cause of acute lower respiratory tract infection (ALTRs) in infants, elderly and immunodepressed patients. Rapid diagnosis of Respiratory Syncytial Virus (RSV) infection is necessary to efficient treatment, avoiding the unnecessary use of antibiotics and determining patient isolation requirements. The reverse trancriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been referred as important tools for virus detection considering the high sensitivity and specificity, respectively of such methods. In order to maximize the simplicity and minimize the risk of sample cross-contamination by two steps RT-PCR, we developed a RT-PCR using a single-tube to detect HRSV in clinical samples. Nasopharyngeal aspirates (Nas) of 226 patients with acute respiratory illness, ranging 0-5 years old, were collected at the University of São Paulo Hospital (HU-USP) in São Paulo city. Samples were tested by indirect immunofluorescence assay, RT semi Nested PCR and single-tube RT-PCR. One hundred two (45,1%) of the 226 samples were positive at least by one of the three methods tested and 75 (33,2%) were positive by all methods. Three (1,3%) samples were positive only by IFI and RT semi Nested PCR, 1 (0,4%) sample were positive only by IFI and RT-PCR single-tube, 5 (2,2%) were positive only by IFI, 2 (0,9%) were positive only by RT semi Nested PCR and 16 (7,1) were positive only RT semi Nested PCR and RT-PCR single-tube. RT-PCR single-tube, showed to be fast, sensitive and specific for diagnosis of RSV and the combined use of both methods enhanced HRSV detection.

vÃrus sincicial respiratÃrio humano (VSRh) à o agente viral mais freqÃentemente relacionado a infecÃÃes do trato respiratÃrio inferior em crianÃas menores de dois anos de idade. O VSRh à caracterizado antigenicamente em dois grupos: A e B, e cada grupo apresenta vÃrios subgrupos. A glicoproteÃna G à a principal responsÃvel pela a variaÃÃo antigÃnica inter e intragrupos desse vÃrus. Os objetivos desse estudo foram caracterizar os perÃodos epidÃmicos e a diversidade antigÃnica e genÃmica dos VSRh circulantes em Fortaleza, Cearà â Brasil, durante cinco perÃodos epidÃmicos consecutivos (2004-2008). A imunofluorescÃncia indireta (IFI) foi utilizada para a triagem de VSRh e de todos os vÃrus analisados e para a caracterizaÃÃo antigÃnica dos VSRh. A RT-nested-PCR seguida do seqÃenciamento parcial do gene G foi utilizada para a caracterizaÃÃo gÃnomica dos VSRh. O VSRh foi detectado em 456 das 2885 (15.8%) amostras. O pico dos perÃodos epidÃmicos de VSRh ocorreu nos meses de marÃo a maio relacionado à ocorrÃncia de chuvas. Um total de 282 VSRh (62,8%) foram caracterizados antigenicamente por IFI, sendo 170 VSRhA (60,3%) e 112 VSRhB (39,7%). Ambos os grupos circularam durante todo o perÃodo analisado sendo observado o predomÃnio de A em todos os anos. Um total de 250 VSRh (54,8%) foi submetido à RT-nested-PCR com amplificaÃÃo de 133 e seqÃenciamento de 86. A caracterizaÃÃo genÃmica dos VSRh identificou os subgrupos GA2 e GA5 para o VSRhA e os subgrupos GB3 e BA para o VSRhB. Esses quatro subgrupos co-circularam durante o ano de 2006. Nos anos de 2004, 2005 e 2007 verificou-se a presenÃa dos dois subgrupos de VSRhA. Em 2008 somente o GA2 circulou. Em 2004, 2007 e 2008 somente o subgrupo BA esteve presente. Em 2005 somente o GB3 circulou. Os VSRhA apresentaram uma maior variabilidade nas seqÃÃncias nucleotÃdicas, indicando uma possÃvel pressÃo seletiva positiva. Houve variaÃÃes no Ãnicio, fim e duraÃÃo de cada perÃodo epidÃmico de VSRh, assim como na circulaÃÃo de grupos e subgrupos. / The human respiratory syncytial virus (HRSV) is the major agent of lower respiratory tract in children under two years old. HRSV is characterized antigenically into two groups: A and B, and each group has several subgroups. Glycoprotein G is primarily responsible for the antigenic variation between and within groups of viruses. The aims of this study were to characterize the epidemic periods and the antigenic and genomic diversity of circulating HRSV in Fortaleza, Cearà - Brazil, for five consecutive epidemic periods (2004-2008). The screening of positive samples to HRSV and other viruses analyzed, as the antigenic characterization of HRSV was carried out by indirect immunofluorescence. RT-nested-PCR followed by partial sequencing of the gene G was used for genomic characterization of HRSV. The HRSV was detected in 456 of 2885 samples (15.8%). The peak of the epidemic periods of HRSV occurred from March to May related to rainfall. A total of 282 HRSV (62.8%) were characterized antigenically, with 170 HRSVA (60.3%) and 112 HRSVB (39.7%). Both groups circulated throughout the period analyzed with a predominance of HRSVA in all years of study. A total of 250 HRSV (54.8%) were submitted to RT-nested-PCR with amplification of 133 and sequencing of 86. The genomic characterization of HRSV identified subgroups GA2 and GA5 for HRSVA and subgroups GB3 and BA for HRSVB. In the years 2004, 2005 and 2007 both subgroups of HRSVA circulated. In 2008 only GA2 circulated. In 2004, 2007 and 2008 only the subgroup BA was present. In 2005 only the GB3 circulated. The HRSV A showed a higher variability in nucleotide sequences, indicating a possible positive selective pressure. There were variations in the beginning, end and duration of each epidemic period of HRSV, as well as in the occurrence of groups and subgroups.

Human respiratory syncytial virus (HRSV) is a negative sense, single strand RNA virus that causes respiratory tract infection with common cold-like symptoms, which can be severe in children, immunocompromised, and the elderly. Even with 60 years of research, the need for vaccine and effective treatment has not been met. In this work, recombinant viruses have been generated which will be valuable in gaining a better understanding of HRSV subtypes, glycoprotein evolution, and the polymerase localization, which would contribute to HRSV vaccine and therapeutics development. The differences in the fitness of A and B antigenic subtypes of HRSV and how it affects the regional circulation pattern is not well understood. To study and compare the two subtypes, it is important to use clinically relevant recombinant viruses and to use animal models that best represent human infection. Using a wild-type virus strain (A11 and B05) from each HRSV subtype, a wild-type like recombinant (r) virus, rHRSVA11, and recombinant viruses expressing fluorescent proteins, rHRSVA11EGFP(5) and rHRSVB05dTom(5), were generated. Characterization of rB05 viruses demonstrated that the differences in the fluorescent protein expressed did not affect virus growth kinetics. To prepare for an experiment in cotton rats, recombinant HRSVs generated were used to infect cotton rat lung cells in vitro. With confirmation of infection of cotton rat lung cells by rHRSV, cotton rat co-infection experiment was planned for the recombinant A11 and B05 viruses and a microneutralization assay was developed for post-infection processing of the in vivo samples. The BA genotype of HRSV B subtype is a strain of HRSV B subtype containing a 60 nucleotide duplication in the glycoprotein (G) gene. HRSV BA genotype was first isolated in 1998 and has quickly become the predominant genotype circulating globally. Although a role of immune evasion by the strains of BA genotype has been suggested to explain this phenomenon, few studies have supported this hypothesis. To compare the HRSV B subtype virus with and without the duplication, rB05 virus lacking the duplication, rHRSVB05EGFP(5)GΔ60b, and containing an epitope tag within the duplication, rHRSVB05EGFP(5)Gmycb, were generated. A serial passage experiment was set up using rHRSVB05EGFP(5) and rHRSVB05EGFP(5)GΔ60b to understand the mutations that accumulate in the G protein gene of each virus. This will be valuable in setting up a similar experiment in the presence of immune pressure to understand the advantage that is conferred to the virus containing the duplication. Expression of Gmyc was confirmed in rHRSVB05EGFP(5)Gmyc infection, which validated that this virus can be used to study the HRSVB05 G protein and modifications in the duplicated region. The HRSV large (L) protein is essential in HRSV transcription and replication, but is difficult to study due to lack of immunologic reagents and challenges with purification. Recombinant viruses expressing reporter and polymerase fusion proteins have been generated and used for studying various other viral polymerases. Expression plasmids for HRSV L protein containing a reporter protein in its variable region 2 have been published. However, the modification resulted in downregulation in the function of the protein and rHRSV expressing modified L protein have not yet been published. In this study, rHRSVB05LVenus was generated to study the effects of modification of HRSV L protein variable region and the localization of HRSV L protein. LVenus protein in rHRSVB05LVenus infected cells was visualized by confocal laser scanning microscopy and the expression levels were examined by immunoblotting. rHRSVB05LVenus was compared to rHRSVB05EGFP(5) with unmodified L protein to show that modification of HRSV L protein had no effect on virus replication. Viruses had equivalent growth kinetics and were equally sensitive to ribavirin, a known HRSV inhibitor. The recombinant viruses generated in this study are valuable tools in answering questions that are difficult to pursue without clinically relevant recombinant viruses. Characterization of the rHRSVs demonstrated that these viruses will have many applications. In this study, viruses were characterized for the basic growth kinetics, expression of proteins of interest, and assay development. With these validated tools, questions such as the cause of the epidemiological pattern observed for HRSV A and B subtypes, the role of host immune response in advantage conferred to HRSV BA genotype, and the effects of inhibitors to formation of HRSV polymerase complex can be addressed. / 2018-10-31T00:00:00Z

The airway epithelium has many roles in innate immunity including detection of pathogens and transmitting danger signals to other cell types. However, its role as a primary defender against infection is not well recognized. We have investigated methods of augmenting antiviral immunity by application of agents that stimulate viral killing, either in the extracellular space or within the cytoplasm. A recently described property of airway epithelial cells is direct oxidative killing of bacteria through the coordination of Duox and lactoperoxidase enzymes. We have exploited this property by supplementing airway cells with the lactoperoxidase substrate iodide to prevent viral infection. A second method for enhancing antiviral defenses is to supply small interfering RNAs (siRNAs) targeting essential viral genes. We have optimized antiviral siRNAs targeting respiratory syncytial virus by designing them to specifically target positive sense viral RNAs. Finally, we have initiated a project to discover host defense genes that are expressed in either the submucosal glands surface epithelium of human airway. This information will enable a better characterization of the roles for these structures in host defense pathways, and may identify other targets for augmentation of antiviral immunity.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection and hospitalization in infants and young children, but no vaccine is currently available. CD4 and CD8 T cells are critical for mediating viral clearance but also contribute to immunopathology following an acute RSV infection. However, few RSV-derived CD4 and CD8 T cell epitopes in the commonly used C57BL/6 mouse strain have been described. I utilized an overlapping peptide library spanning the entire RSV proteome and intracellular cytokine staining for interferon-gamma (IFN-γ) to identify novel CD4 and CD8 T cell epitopes in C57BL/6 mice. I discovered and characterized two novel CD4 T cell epitopes and three novel CD8 T cell epitopes located within multiple RSV proteins. Overall, the novel RSV-derived CD4 and CD8 T cell epitopes identified in C57BL/6 mice will aid in future studies of RSV-specific T cell responses. While CD8 T cells are important for viral clearance following an acute RSV infection, the contribution of memory CD8 T cells in providing protection against reinfection with RSV remains unclear. I used a prime-boost immunization approach to induce robust, systemic memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. I determined that high magnitude, systemic memory CD8 T cell responses efficiently reduced lung viral titers following RSV infection, but unexpectedly did so at the expense of severe and fatal immunopathology. The exacerbated disease was mediated by the rapid and excessive production of IFN-γ by memory CD8 T cells in the lung and airways. In contrast, I found that local immunization generated a large population of tissue-resident memory CD8 T cells in the lung that efficiently reduced lung viral titers in the absence of exacerbated disease. Additionally, I observed that pre-existing RSV-specific neutralizing antibodies prevented the immunopathology induced by high magnitude, systemic memory CD8 T cell responses following RSV infection. Prophylactic treatment with neutralizing antibodies against RSV efficiently restricted early virus replication, which resulted in a significant decrease in lung IFN-γ levels, memory CD8 T cell activation, and the frequency of IFN-γ producing CD8 T cells. Thus, my results demonstrate that high magnitude, systemic memory CD8 T cells induce lethal immunopathology following RSV infection, which can be prevented by pre-existing RSV-specific neutralizing antibodies. Overall, my results have important implications for the development of future RSV vaccines. The development of a live-attenuated vaccine for RSV has been prevented by the inability to properly balance attenuation with immunogenicity and efficacy. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. As the M protein is required for virion assembly following infection of a host cell, RSV M-null induces a single-cycle infection. I evaluated RSV M-null as a potential live-attenuated vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared to its recombinant wild-type control virus (RSV recWT). RSV M-null was sufficiently attenuated, as significantly reduced lung viral titers, weight loss, and pulmonary dysfunction were observed compared to mice infected with RSV recWT. Surprisingly, despite its attenuation, I found that RSV M-null infection induced effector T cell, germinal center B cell, serum antibody, and memory T cell responses of similar magnitude to that elicited by infection with RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, my results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis-induced hospitalization in young children. A natural RSV infection fails to elicit long-lasting immunity, further increasing the need for an effective vaccine. Despite the significant healthcare burden, there is no licensed RSV vaccine currently available. While most RSV vaccine strategies focus on the induction of humoral immunity, high antibody titers do not prevent RSV infection. It remains unclear if protective immunity can be achieved through robust cellular immunity. Previous work has indicated that a relatively low frequency of virus-specific CD8 T cells is induced following an RSV infection in human infants. In addition, RSV-specific memory CD8 T cells diminish to almost undetectable frequencies in the blood of the elderly. The lack of long-lasting immunity against RSV may be explained by an absence or low frequency of memory CD8 T cells within the lung following infection. However, I determined that the majority of effector CD8 T cells reside within the lung tissue following infection with either RSV or influenza A virus (IAV), both of which replicate primarily in the airways. In addition, approximately 70% of antigen-experienced memory CD8 T cells persist in the lung tissue at day 30 following RSV infection. In contrast, the majority of CD8 T cells remain in the pulmonary vasculature following intranasal infection with either of the systemically replicating viruses lymphocytic choriomeningitis virus or vaccinia virus. Therefore, the tissue tropism of a virus will determine if CD8 T cells preferentially accumulate in the lung tissue following infection of the respiratory tract. An experimental formalin-inactivated RSV (FI-RSV) vaccine caused enhanced respiratory disease in vaccinated children following a natural RSV infection. Incomplete knowledge of the underlying immunological mechanisms that were responsible for mediating the enhanced disease has greatly hampered vaccine development. Previous studies have indicated that eosinophils, non-neutralizing antibodies, and CD4 T cells may be required to elicit FI-RSV vaccine-enhanced disease. I determined that distinct CD4 T cell subsets mediate individual disease parameters. The Th2-biased immune response, but not eosinophils specifically, was responsible for induction of airway hyperresponsiveness and mucus hypersecretion. On the other hand, the Th1-associated pro-inflammatory cytokine TNF-α was required to mediate baseline pulmonary dysfunction and weight loss. Lastly, while depletion of CD4 T cells ameliorated all disease parameters evaluated, the antibody titers remained unaltered in depleted mice. Thus, antibodies induced by FI-RSV immunization were not required for vaccine-enhanced disease. My data demonstrate that discrete disease manifestations associated with FI-RSV immunization are orchestrated by distinct subsets of CD4 T cells. The CD8 T cell response is believed to contribute to both pathogen clearance and immunopathology following an acute RSV infection. However, it is unclear if robust memory CD8 T cell responses will protect against an RSV infection. I determined that induction of a high-magnitude, epitope-specific memory CD8 T cell pool mediated increased viral clearance following RSV challenge. However, mice with robust secondary CD8 T cell responses exhibit increased airway dysfunction, weight loss, and mortality as compared to mock-immunized mice undergoing an acute RSV infection. The enhanced disease severity was unique to the context of an RSV infection as similarly immunized mice were protected from chge with a lethal dose of a recombinant IAV engineered to express an RSV-derived epitope. In addition, the increased morbidity and mortality was associated with an elevated amount of both IFN-γ and TNF-α in the serum of immunized mice. Neutralization of either IFN-γ or TNF-α led to a significant reduction in disease severity and survival of all mice. These results demonstrate that robust memory CD8 T cell responses enhance viral clearance, but also lead to severe pulmonary immunopathology following RSV infection. Overall, I establish that the majority of effector CD8 T cells are localized within the lung tissue following a respiratory infection, and determine that either memory CD4 or CD8 T cell responses elicits severe immunopathology following a RSV infection.

During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.

Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 42-47).