Polymerase basic protein 1 (PB1) as a molecular determinant of fitness and adaptation in influenza a virus

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2017The World Health Organization and the National Institute of Allergy and Infectious Diseases reported growth deficits of influenza A(H1N1)pdm09 reverse genetic pandemic vaccine virus seeds. These have compromised the effective and timely distribution of vaccines in the 2009 pandemics and accentuated the need to improve the process of vaccine production. In pre-pandemic A(H5N1) research, seed viruses produced by reverse genetics have also been reported to present growth deficits. These deficits have been attributed to a putative sub-optimal protein interaction. The dynamics of the genetic evolution of influenza A viruses appears to suggest a gene segregation pattern between the Polymerase Basic protein 1 (PB1) and antigenic proteins Hemagglutinin (HA) and Neuraminidase (NA). In the reassortment events that lead to the emergence of the 1957 e 1968 pandemic viruses, the contemporary seasonal viruses acquired PB1 genomic segment together with antigenic glycoproteins originating from avian viruses. A similar pattern was identified in 1947, where a reassortment event between seasonal viruses, involving PB1 and antigenic proteins, has altered the epidemiology of the infection to a near-pandemic geographic dispersion. In both situations, viral fitness appears to have benefitted from acquiring a PB1 genomic segment homologous to antigenic proteins. Also, in retrospective studies on the genomic composition of high yield seasonal vaccine seeds produced by classical reassortment, PB1 is frequently co-incorporated with antigenic proteins HA and NA, further suggesting that the interaction between these proteins could have an impact in viral fitness. In this context, we proposed to address the question of PB1 genomic segment being a molecular determinant of fitness and adaptation in influenza A virus and, particularly, of the functional compatibility between PB1 and antigenic proteins being a driver of the overall viral fitness and putatively exploitable to improve seed virus production. The A(H1N1)pdm09 virus was used a model for this research because it is a product of viral reassortment with an unprecedented genomic composition of segments originating from avian, swine and human seasonal viruses. Additionally, the 2009 pandemic vaccine virus presented severe growth deficits and, since the A(H1N1)pdm09 persists in circulation with a seasonal epidemiologic profile, the demand for high yield A(H1N1)pdm09 vaccine seeds will be continuous and the need to adequate the immunogenic strain to the circulating viruses will be recurrent because of antigenic drifts. The objectives of this research were defined as 1) to evaluate the genetic evolution of PB1 in the zoonotic transmission of swine influenza virus and infer its putative contribution towards viral fitness and adaptation, and 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetics A(H1N1)pdm09 vaccine seed model. The approach followed to accomplish objective 1 was to select a study sample of PB1 nucleotide sequences from swine virus that have infected the human host, to analyze phylogeny and mutation trends and to search for putative markers for viral adaptation on the basis of viral molecular epidemiology, genomic location of the polymorphisms and amino-acid properties. Our major findings were that the evolutionary history of PB1 is traceable in terms of lineage and host origin. Specific genomic markers in PB1 appear to putatively relate to the viral adaptation to mammalian hosts, 336I, 361R, 468K and 584Q, and to the viral adaptation to new genomic backgrounds possibly in the sequence of reassortment events, such as 638D and 618D. Residues 298I, 386K and 517V have been found to putatively relate to an enhanced compatibility between PB1 and HA of the H1 subtype, in the mammalian host. A subsequent in vitro investigation of the phenotypic impact of mutations L298I, R386K and I517V acquired by the A(H1N1)pdm09 during its evolutionary history, was performed by generating an A(H1N1)pdm09 recombinant virus and an A(H1N1)pdm09 reassortant in which the specific mutations have been reverted, by reverse genetics. This approach has resulted in two major findings. Acquiring these mutations has been found to putatively promote conformational changes in PB1 and enhance the span of complementary nucleotides possibly involved in PB1 interaction with HA at the RNA level and, on the other hand, has proven detrimental to viral growth kinetics in vitro. These findings have lead us to suggest that the interaction between genomic segments at the RNA level could be a determinant of co-segregation, concordant with a selective packaging model proposed by other authors, but that the mechanisms that drive this process are probably not dependent on a replicative advantage. Our approach to accomplishing objective 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetic A(H1N1)pdm09 vaccine seed model, was to determine the genetic profile of A(H1N1)pdm09 strains circulating in Portugal during the pandemic period and select a prototype immunogenic strain, to generate reassortant viruses with the genomic composition of A(H1N1)pdm09 seed viruses prototypes bearing PB1 homologous and heterologous to antigenic proteins, and to evaluate viral growth and antigen yield in vitro. A sample of specimens collected from the pandemic period in Portugal were evaluated for genetic and phenotypic features and a strain similar to the consensus was selected as a prototype strain. Vaccine seed prototypes of the selected A(H1N1)pdm09 strain in an A/PuertoRico/08/34 backbone were generated by reverse genetics to present the genomic compositions of the 6:2 classical vaccine seed (PR8:HA,NA A(H1N1)pdm09) and a 5:3 seed prototype in which the PB1 segment from the immunogenic strain is co-incorporated with the antigenic proteins (PR8:HA,NA,PB1 A(H1N1)pdm09). Our major findings were that the presence of PB1 homologous to antigenic protein significantly increased viral replication, hemagglutination capacity and Neuraminidase activity. We have establishing proof of concept that, in the PR8:A(H1N1)pdm09 seed virus model, viral growth and antigen yield can be significantly improved by the inclusion of PB1 from the immunogenic strain when compared to the classical seed virus prototype. We consider that, additionally to the role of PB1 protein in viral replication, PB1 genomic segment may be a molecular determinant of the overall virus fitness and a determinant factor in the molecular epidemiology of the viruses by establishing interactions with other segments at the RNA level and by, apparently, being able to genetically change and adapt to improve these interactions. Further research is necessary to clarify the mechanisms of viral genome packaging, the role of interactions at the RNA level in establishing the co-segregation patterns and the specificities of this interactions at the subtype level. However, it becomes clear that the functional compatibility between PB1 and antigenic proteins is a driver of the overall viral fitness in the A(H1N1)pdm09 and is putatively exploitable to improve seed virus production. We also consider that exploring the concept of the compatibility between gene segments or proteins being a determinant factor in the overall viral fitness, can result in major improvements in the production of reverse genetics seed viruses of different influenza subtypes. Also, being aware of the fact that the genomic composition of influenza viruses can have a major phenotypic impact, and that consequently is a determinant of virulence even though the mechanisms that drive the selective packaging remain unclear, we consider that its inclusion in the risk assessment of influenza strains would be extremely relevant for seasonal and pandemic preparedness

Os conhecimentos fortuitos no contexto das buscas domiciliárias: da relevância dos conhecimentos fortuitos e sua valoração

Os meios de obtenção de prova apresentam-se como métodos eficazes na recolha dos meios de prova, os quais, posteriormente apreciados pela Autoridade Judiciária (AJ) competente no processo, poderão ser determinantes na descoberta da verdade. Os conhecimentos fortuitos, fonte de informações análogas a um crime que não o de investigação, surgiram no âmbito das intercepções telefónicas, tendo sido na Alemanha que se iniciou a abordagem sobre esta problemática. Com a entrada em vigor da Lei 48/2007, os conhecimentos fortuitos passam a estar tipificados no ordenamento jurídico português, colmatando as críticas que se ostentavam quanto à necessidade de reserva de lei sobre os conhecimentos adversos. Deste tema, a diversa doutrina encara três posições quanto ao modo de utilização dos conhecimentos fortuitos, desde a valoração absoluta, a recusa total de valoração e a valoração condicional. Contudo, não apenas nas escutas telefónicas, os investigadores têm conhecimento de outros factos que não se enquadram no objecto da investigação. Uma vez que os Órgãos de Polícia Criminal (OPC) têm um papel essencial na descoberta de meios de prova, o tratamento a ser executado quanto aos conhecimentos fortuitos não poderá suscitar qualquer dúvida, implicando uma intervenção urgente para que os meios de prova não sejam dispersos

Study of mortality by Diabetes Mellitus in Portugal

A diabetes mellitus (DM) é considerada um dos problemas de saúde pública de maior importância a nível mundial, pela elevada prevalência, morbilidade e mortalidade (Sousa, 2006), prevendo-se um agravamento na próxima década relacionado com o envelhecimento da população e com as alterações progressivas no estilo de vida (Cruz, 2005). A Organização Mundial de Saúde (WHO) estima que a diabetes possa vir a ser responsável pela primeira regressão na esperança média de vida dos últimos 200 anos. Pretende-se, com o presente estudo, avaliar a evolução temporal e a distribuição geográfica da mortalidade por DM em Portugal

Do Consentimento nas Buscas Domiciliárias: Contributos para a valoração da prova

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Influenza A(H1N1)pdm09 Resistance and Cross-Decreased Susceptibility to Oseltamivir and Zanamivir Antiviral Drugs

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011. A total of 144 cases of A(H1N1)pdm09 virus infection from community and hospitalized patients were studied, including three suspected cases of clinical resistance to oseltamivir. Oseltamivir resistance was confirmed for two of the suspected cases. Neuraminidase (NA) H275Y resistant marker was found in viruses from both cases but for one it was only present in 26.2% of virus population, raising questions about the minimal percentage of resistant virus that should be considered relevant. Cross-decreased susceptibility to oseltamivir and zanamivir (2-4 IC50 fold-change) was detected on viruses from two potentially linked community patients from 2009. Both viruses harbored the NA I223V mutation. NA Y155H mutation was found in 18 statistical non-outlier viruses from 2009, having no impact on virus susceptibility. The mutations at NA N369K and V241I may have contributed to the significantly higher baseline IC50 value obtained to oseltamivir for 2010/2011 viruses, compared to viruses from the pandemic period. These results may contribute to a better understanding of the relationship between phenotype and genotype, which is currently challenging, and to the global assessment of A(H1N1)pdm09 virus susceptibility profile and baseline level to NAIs

o contributo dos homens de confiança para a produção de prova e a sua perigosidade

The production of evidence is the central object of criminal investigation. Finding out the existence of a crime and determining its agents, allowing the discovery of the truth, prosecution of the guilty and the acquittal of the innocent is the basis for investigation of any wrongdoing. The reconstitution of a fact is not always intelligible, so investigators must take into account all versions that arise on the criminal scenario. They should take into account that in addition to their investigation training, there are people who get information about a reality that is sometimes only possible to reach with their testimony. The use of this gathered information can be a key catalyst for the accountability of perpetrators. However, investigators should be aware of how this information was obtained. The very relationship between the police officer and the human source of information must respect certain principles and rules so that the main subject of the criminal investigation is not accused under deceptive and offensive means against its rights, freedoms and guarantees. In addition to the human sources of information, there are other evidence collection entities that some authors designate as reliable men. They all aim at identifying means and methods that can contribute to the investigation, but the line between legality and illegality is very thin, so there should be restraint on creating criminal scenarios that could compromise the legality of the investigation.A produção de prova apresenta-se como o objecto central da investigação criminal. Apurar a existência de um crime e determinar os seus agentes, permitindo a descoberta da verdade, acusação dos culpados e ilibação dos inocentes é a base de investigação de qualquer ilícito. Nem sempre a reconstituição de um facto é inteligível, daí que os investigadores tencionarão ouvir as vozes que clamam no cenário criminal. Devem atender que para além da sua formação investigatória, existem pessoas que obtém informação sobre uma realidade que por vezes só é possível atingir com o seu testemunho. A utilização das informações recolhidas pode ser um catalisador fulcral para a responsabilização dos autores criminais. Porém, os investigadores deverão perceber como essa informação foi recolhida. A própria relação entre o polícia e a fonte humana de informação deve respeitar determinados princípios e normas para que o objecto principal da investigação criminal não seja cumprido sob meios enganosos e ofensivos aos direitos, liberdades e garantias. Para além das fontes humanas de informação, existem outras figuras de recolha de prova a que alguns autores designam por homens de confiança. Todas visam a identificação de meios e métodos que contribuem para a investigação, mas a linha que separa a legalidade da ilicitude é muito estreita, devendo os meios na dependência do Estado se abster da criação de cenários criminais que possam comprometer a legalidade da investigação

Modified kraske procedure with mid-sacrectomy and coccygectomy for en bloc excision of sacral giant cell tumors.

Sacral giant cell tumors are rare neoplasms, histologically benign but potentially very aggressive due to the difficulty in achieving a complete resection, their high recurrence rate, and metastization capability. Although many treatment options have been proposed, en bloc excision with tumor-free margins seems to be the most effective, being associated with long term tumor control, improved outcome, and potential cure. An exemplifying case of a 29-year-old female with progressive complaints of pain and paresthesias in the sacral and perianal regions, constipation, and weight loss for 6 months is presented. The surgical technique for en bloc excision of a large sacral giant cell tumor through a modified Kraske procedure with mid-sacrectomy and coccygectomy is described. Complete resection with wide tumor-free margins was achieved. At 5 years of follow-up the patient is neurologically intact, without evidence of local recurrence on imaging studies. A multidisciplinary surgical procedure is mandatory to completely remove sacral tumors. In the particular case of giant cell tumors, it allows minimizing local recurrence preserving neurovascular function, through a single dorsal and definitive approach

Outbreak of acute respiratory infection among infants in Lisbon, Portugal, caused by human adenovirus serotype 3 and a new 7/3 recombinant strain

Human adenoviruses (AdVs) typically cause mild illnesses in otherwise healthy hosts. We investigated a pediatric outbreak of acute respiratory infection with fatal outcomes that occurred in Lisbon, Portugal, in 2004. Biological specimens were collected from 83 children attending two nurseries, a kinesiotherapy clinic, and the household of a nanny. Adenovirus infection was confirmed in 48 children by PCR and virus isolation. Most (96%) isolates were classified as being of subspecies B1. Phylogenetic analysis of fiber and hexon gene sequences revealed that most infants were infected with AdV serotype 3 (AdV3) strains. Infants attending one nursery harbored a new recombinant strain containing an AdV serotype 7 hexon and serotype 3 fiber (AdV7/3). Both the AdV3 and the AdV7/3 strains caused fatal infections. Two different serotype 3 strains were circulating in Lisbon in 2004, and the new AdV7/3 recombinant type originated from only one of those strains. These results demonstrate that recombination leads to the emergence of new adenovirus strains with epidemic and lethal potential

COMPARAÇÃO DA CIRURGIA CONVENCIONAL COM A LAPAROSCÓPICA NO CANCRO DO RETO: RESULTADOS DO REGISTO PORTUGUÊS

Introduction: This study aimed to compare the 3-year rates of local recurrence (LR) and overall survival (OS) for open (OPEN) and laparoscopic (LAP) surgeries in a Portuguese registry. Material and Methods: This observational study included patients who underwent rectal cancer resection performed in 16 hospitals between July 2014 and December 2019. The radiologic staging and the specimen images of the first three cases of any hospital were uploaded and audited by the scientific committee. Clinical and pathological characteristics and short and long-term outcomes of OPEN and LAP surgeries were analyzed. Results: The registry included 640 patients who underwent rectal cancer surgery: 562 (87.8%) underwent curative resection and 78 (12.2%) underwent palliative resection. In the curative cohort, OPEN surgery was performed in 269 cases whereas LAP surgery, which had a conversion rate of 17.5%, was performed in 266 cases. The pN staging showed that the LAP group had less advanced disease than the OPEN group. Anterior resection was performed in 57.8% of the cases whereas abdominoperineal resection was performed in 16.5%. Patients who underwent LAP surgery had shorter hospital stays. The 3-year LR rate was 3.0% (95% CI, 1.4%-6.3%) for LAP surgery and 8.3% (95% CI, 5.1%-13.1%) for OPEN surgery (P=0.02). The 3-year OS was 88.2% (95% CI, 83.1%-92.0%) for LAP surgery and 76.5% (95% CI, 69.1%-82.6%) for OPEN surgery (P=0.0061). Discussion: LAP surgery for patients with rectal cancer is associated with a decreased LR rate and improved OS, although in those with less advanced pN staging. Conclusion: The data support the view that the LAP approach is justified for rectal cancer when performed by surgeons with appropriate laparoscopic experience.Introdução: O objetivo do estudo consistiu na avaliação da recidiva local (RL) e da sobrevivência global (SG) aos 3 anos, comparando cirurgia convencional (CONV) e laparoscópica (LAP) no registo Português do cancro do reto. Material e Métodos: Neste estudo observacional incluíram-se doentes com cirurgia por cancro do reto realizada em 16 hospitais, entre Julho 2014 e Dezembro 2019. O estadiamento imagiológico e as imagens anatomopatológicas foram registadas e auditadas pela comissão científica. Analisaram-se as características clinico-patológicas e os resultados pós-operatórios e à distância na cirurgia CONV e LAP. Resultados: O registo inclui 640 doentes que realizaram cirurgia por cancro do reto: 562 (87.8%) resseções curativas e 78 (12.2%) resseções paliativas. No grupo curativo foram realizadas 269 resseções CONV e 266 resseções LAP, que tiveram conversão em 17,5% dos casos. O grupo LAP tinha estadiamento pN menos avançado que o grupo CONV. A resseção anterior foi realizada em 57,8% dos casos e a amputação abdominoperineal em 16,5%. Os doentes com cirurgia LAP tiveram estadia pós-operatória mais curta. A taxa de RL aos 3 anos foi de 3,0% (95% CI, 1,4%-6,3%) na cirurgia LAP e 8,3% (95% CI, 5.1%-13,1%) na cirurgia CONV (P=0.02). A SG aos 3 anos foi 88,2% (95% CI, 83,1%-92,0%) na cirurgia LAP e 76,5% (95% CI, 69,1%-82,6%) na cirurgia CONV (P=0.0061). Discussão: Nos doentes com cancro do reto a cirurgia LAP associou-se a menor taxa de RL e melhor SG, embora em doentes com estadiamento pN menos avançado. Conclusão: Estes resultados confirmam que no cancro do reto a abordagem LAP é segura se for realizada por cirurgiões com adequada experiência laparoscópica