No mês de março de 2013 foram reportados pela Organização Mundial da Saúde (OMS) os primeiros casos de infecções humana pelo vírus Influenza aviário A(H7N9). Em maio de 2017 já haviam sido registrados 1439 casos. As infecções em humanos causadas por este vírus são associadas a consideráveis níveis de mortalidade e morbidade. A vacinação é atualmente o meio mais efetivo para prevenir as infecções e as severas complicações na saúde humana causada pelo vírus da Gripe. Desenvolver e produzir vacinas contra a Gripe pandêmica é a estratégia principal para uma resposta contra a pandemia (WORLD HEALTH ORGANIZATION, 2005). Este projeto produziu os bancos de vírus H7N9, um lote piloto e três lotes industriais em condições (BPF) dos monovalentes utilizados para a produção da vacina contra a gripe pandêmica, A(H7N9). Estes lotes foram caracterizados, avaliados em relação aos padrões de qualidade e testados quanto a imunogenicidade em ensaios com camundongos. / In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). In May 2017 they had been registered 1439 cases. The infections in humans caused by this virus are associated with considerable morbidity and mortality levels. Vaccination is currently the most effective way to prevent infections and severe complications on human health caused by the flu virus. Developing and producing vaccines against pandemic influenza is the main strategy for a response to the pandemic (WORLD HEALTH ORGANIZATION, 2005). This project produce the seeds virus A(H7N9), a pilot batch and three industrial lots (GMP) of the monovalents used to produce the pandemic influenza vaccine against A(H7N9). These batches were characterized, evaluated for quality standards and tested for immunogenicity in mouse assay.

Influenza viruses cause significant mortality and morbidity worldwide due to seasonal oubreaks as well as occassional, and sometimes devastating, pandemics. Estimates state that approximately 5% of the adult and 20% of the child population is infected yearly, leading to approximately a half-million deaths and three million severe infections in non-pandemic years. Aside from globally-circulating strains, zoonotic outbreaks caused by avian strains are a constant threat. In 1997, the first human cases of H5N1 infections occurred and since then strains of this subtype have killed approximately 700 people causing a severe disease with as high as 60% lethality rate. In March 2013, a strain of the H7N9 subtype started an epizootic in China causing a severe respiratory disease reminiscent of H5N1 infections and with a 20% case fatality rate. In this thesis, we have studied the host responses as well the viral replication kinetics of H5N1 and H7N9 strains and compared then to those of mild H1N1 seasonal and 2009 pandemic strains. During early infections of A549 cells, we have shown that the H5N1 virus induced a more profound and functional change to the host proteome. All viruses induced the NRF2-mediated oxidative stress responses and the H7N9 and H5N1 strains downregulated fibronectin, a host protein vital to infection for human strains. Using mathematical modeling and extensive growth kinetic analysis, we showed that the H5N1 and H7N9 strains had higher peak titers and faster growth kinetics. This was due to an higher infection rate for the H7N9 strain and an higher production rate for the H5N1 strain, compared to the human viruses. Conversely, the 2009 pandemic H1N1 strain had the poorest replication kinetics, longest eclipse phase and lowest infection rates. These results point towards the higher level of cellular disruption during infection with highly pathogenic strains of influenza, which may be indicative of the more profound changes required to support growth of viruses with faster kinetics to higher titers. Furthermore, the greater changes in the cellular proteome that we have characterized in vitro may be connected to the significantly greater virulence associated with infection by avian viruses in vivo, opening a novel and productive avenue of investigation to understand viral virulence mechanisms. / October 2015

Influenza is a viral infection that affects global health and economy with its endemic and sometimes pandemic spread. Rapid detection of Influenza viruses enables antiviral use and can bring financial savings. It is also essential for the global surveillance of prevalent Influenza strains. RT-PCR is considered the most specific and sensitive method for detection of Influenza, but Influenza mutates at a high rate and it is therefore crucial that RT-PCR methods are updated regularly. In 2014, Cepheid released their Xpert Flu/RSV XC assay, which can detect Influenza A and B and RSV by multiplex RT-PCR in approximately one hour. The aim of this study was to evaluate this assay at Laboratoriemedicin Västernorrland by using the laboratory’s previous PCR assay for detection of Influenza viruses as reference method. Real-time RT-PCR was used to compare Xpert Flu/RSV XC to the reference method. A dilution series was performed to estimate the methods’ PCR efficiencies and precision was calculated from quadruplicates of a positive control sample. Clinical specimens (n=42) were used to evaluate the diagnostic sensitivity and specificity of Xpert Flu/RSV XC. Objective statistical analysis of PCR data was performed and discussed. The Xpert Flu/RSV XC was equivalent to the reference method and demonstrated high diagnostic sensitivity and specificity. Estimated PCR efficiencies were however low. With the introduction of Xpert Flu/RSV XC to the laboratory follows many potential benefits, primarily in form of a simplified pre analytical procedure and a shortened analysis time. The Xpert Flu/RSV XC assay enables fast diagnosis of Influenza infection.

What explains the geographic distribution of pathogens? Better understanding and characterising disease patterns will help scientists to identify areas likely to host future epidemics and epizootics and to prioritise surveillance and intervention. However, the use of disease surveillance data to assess the risk of transmission and generate risk maps raises conceptual and methodological issues. Indeed, pathogens and more particularly viruses aren't ”species” like others that live in the open environment and must be studied with methods and concepts of their own. Avian influenza (AI), a disease caused by a virus infecting bird populations, has been selected to study these issues. AI has a major economic impact on the poultry industry in many countries, raises concerns of livelihood in low and middle-income countries, and represents a major concern for human health. The aim of this PhD thesis was to improve the knowledge on the spatial epidemiology of AI in different settings and conditions (i). For this, recent epizootics caused by the subtypes A (H5N1) and A (H7N9) were selected as case studies. First, highly pathogenic subtypes of the A (H5N1) virus have been studied in poultry farms (ducks and chickens) at different spatial scales: at the continental scale and the regional scale in the Mekong (Cambodia, Laos, Vietnam, Thailand) and the Nile Delta in Egypt. All these cases occurred between 2003, the date on which the virus starts to spread outside China, and 2015; the HPAI A (H5N1) subtypes are still reported today in many countries. Human infections caused by the A (H7N9) virus in China from March 2013 to 2017 were also studied. Studied different AI subtypes at different spatial scales within different host species also allowed to develop a conceptual model of AI transmission and to discuss the issue of the transferability of results in epidemiology (ii). Lastly, this PhD thesis leads to a discussion about the transfer of methods and concepts from ecology to spatial epidemiology, with a particular emphasis on their possible limitations (iii). / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished