Thesis (Ph.D.) - University of St Andrews, May 2008.

Picornaviruses replicate in association with cytoplasmic membranes of infected cells. Poliovirus 2C and 2BC play an important role in the formation of membranous vesicles, and induce dramatic changes in membrane trafficking. Theiler’s murine encephalomyelitis virus protein 2C was localized in infected cells using an anti-TMEV-2C antibody. Early upon infection, TMEV 2C was localized in the cytoplasm in an ER-like pattern. At later stages, 2C redistributed to a juxtanuclear site, which represents the viral replication site. Co-localization with the Golgi complex could not be observed. TMEV 2C seems to interact in vitro with reticulon 3, a highly conserved ER-associated protein. It was not possible to confirm a previously identified interaction with AKAP10, a protein kinase anchoring protein, presumably reflecting conformational constraints of the interaction. Two mutations in the AKAP10 binding site of TMEV 2C were identified, which inhibit the completion of the infectious cycle of TMEV. The intracellular changes that occur during TMEV infection were observed. Both actin filaments and microtubules may be used at early stages of infection; however both cytoskeleton components accumulate at the periphery of the cell during late stages of infection. A computer- based analysis has demonstrated that TMEV 2C is highly similar to katanin, a microtubule- severing protein, and may play a similar role in the reorganization of microtubules during infection. The Golgi complex turns from a solid, crescent-shaped organelle, into a series of punctuate fluorescent points forming an expanding balloon-like structure surrounding the concomitantly expanding site of virus replication. The remnants of the Golgi complex are finally dispersed throughout the cytoplasm. Live imaging confirmed these findings. It was observed that PKA also undergoes displacement to the cell periphery during infection. However, BIG1 seems to locate to the viral replication site during infection, suggesting it may play a role during viral replication. The localization of PKA and BIG1 in the infected cell may in part explain the observed dispersion of the Golgi complex.

Polioviruses have been around for a long time in man's history. Before the development of killed and live virus vaccines in the 1960's, poliomyelitis was a serious problem in public health. Since then paralytic poliomyelitis remains a threat in certain underdeveloped countries but has been considered a conquered disease in the developed world. The molecular epidemiology of wild-type 1 polioviruses (WPV1) isolated in Angola in 2005, the Democratic Republic of the Congo (DRC) in 2006-2008 and Namibia in 2006 were investigated by sequence analysis of the complete VP1 gene of all isolates. All outbreak viruses clustered with the Indian type 1 genotype (SOAS) which was unique to India circulating endemically in the Uttar Pradesh (UP) and Bihar provinces in Northern India. Epidemiological and virological analyses suggested that the Namibia outbreak virus had been circulating without detection for at least one year in Angola. Four cases of acute flaccid paralysis (AFP) occurred in children in Madagascar in 2005. Molecular analysis confirmed cVDPV type 2 and 3 in affected patients. The first case, occurred in Toliara II district, on 09 April 2005. The last two cases were in the Toliara I and Beloha districts and paralysis onset on 26 June and 13 July 2005 respectively. Partial genomic sequencing of the poliovirus isolates revealed considerable divergence from the prototype Sabin strain in all cases. This is the second time that type 2 cVDPV is associated with an outbreak of AFP in Madagascar, and to our knowledge the first time that a type 3 cVDPV is identified in Madagascar. A total of fifty-six children with AFP were found to excrete VDPVs of serotype 2 in the DRC between 2005 and 2010. These viruses represent at least three emergences and at least two outbreaks. Partial genomic sequencing of the poliovirus isolates revealed considerable nucleotide sequence divergence of between 1.1% to 2o/o from the prototype Sabin strain in the VP1 region of the viral genome. This was the first time that a type 2 cVDPV outbreak was detected in the DRC. In total, 89 viral isolates obtained from Ethiopia during 2007 to 2010 and partial sequencing analysis confirmed that 13 isolates were VDPV's. Seven AFP cases were type 3, 4 AFP cases were type 2 and 2 contacts for type 3. Partial genomic sequencing of the poliovirus isolates revealed considerable divergence from the prototype Sabin strain in all cases. Finally, cases of AFP where only Sabin-like viruses were identified were investigated in South Africa with 11 possible VAPP cases identified with recombinant events in the 30 region and also revealing a mutation that restore the original stem-loop structure in the internal ribosomal entry site (IRES) in the 5' Non-Translated Region (NTR). In this study, the molecular epidemiology of poliovirus outbreaks that occurred in Angola, Namibia, and the DRC is described that were associated with wild polio 1 and 3. Investigation of Sabin-like vaccine strains in the DRC, Madagascar and Ethiopia identified vaccine-derived polioviruses in AFP cases as well as possible vaccineassociated paralytic poliovirus in South Africa. Copyright / Thesis (PhD)--University of Pretoria, 2012. / Medical Virology / Unrestricted

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-17T13:17:37Z No. of bitstreams: 1 Hugo Leonardo2 OK.docx.pdf: 1727936 bytes, checksum: 9b422dec3434e2e337a66ba7ab57e26b (MD5) / Made available in DSpace on 2013-09-17T13:17:37Z (GMT). No. of bitstreams: 1 Hugo Leonardo2 OK.docx.pdf: 1727936 bytes, checksum: 9b422dec3434e2e337a66ba7ab57e26b (MD5) Previous issue date: 2012-08-30 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os enterovírus humanos ( Picornaviridae ) são vírus de transmissão predominantemente ent érica, possuem distribuição cosmopolita, estando entre os agentes mais prevalentes como causadores de patogenias em humanos. Atualmente, já foram descritos mais de 100 sorotipos para os enterovírus humanos e em grande parte dos casos as infecções associada s são assintomáticas. Surtos e casos esporádicos de enteroviroses são frequentemente notificados em diversas regiões do mundo causando conjuntivite hemorrágica aguda, meningite asséptica, doença de mão pé e boca e poliomielite. A poliomielite é uma doença infecciosa de caráter agudo, que pode assumir desde formas assintomáticas até formas paralíticas (paralisia flácida aguda ou PFA), causada em geral por um dos três sorotipos de poliovírus (PV). O PV selvagem está eliminado do Brasil desde 1989, atualmente sendo restrito a apenas quatro países (Nigéria, Afeganistão, Paquistão, Índia). Entretanto surtos de PFA associados à PV de origem vacinal e a enterovírus não pólio recombinantes tem sido notificados. A caracterização de EVNP é de extrema importância para a investigação da diversidade de vírus co - circulantes, e para relacionar os sintomas clínicos com o sorotipo viral envolvido, incluindo a investigação de vias de transmissão de enterovírus, durante a ocorrência de surtos, além de contribuir para estudos ep idemiológicos e com a evolução de enterovírus. Neste estudo foram analisadas amostras relacionadas à PFAs, utilizando RT - PCR e PCR com o objetivo de identificar quais são as espécies de enterovírus humanos associadas. Os membros da espécie C foram sequenci ados para a identificação de sorotipo. De um total de 190 amostras, 79 eram da espécie C, 78 da espécie B, 32 da espécie A e 1 amostra era correspondente as espécies A e C,não sendo encontradas amostras da espécie D. Entre as amostras da espécie C,58 cor respondiam a PV.Os dados obtidos apresentam similaridades com estudos similares na Europa e Ásia, cobrindo um aspecto pouco observado na epidemiologia dos enterovírus em território brasileiro. / The human enterovirus es (Picornaviridae) are predominantly enteric virus transmission, have a cosmopolitan distribution, being among the most prevalent agents as causing pathogens in humans. Currently, have been described more than 100 serotypes for human enteroviruses and in mo st cases associated infections are asymptomatic. Outbreaks and sporadic cases of enteroviruses are frequently reported in several regions of the world causing acute hemorrhagic conjunctivitis, aseptic meningitis, hand foot and mouth disease and poliomyelit es. Poliomyelites is an infectious disease of acute character, which may take from asymptomatic to paralytic form (acute flaccid paralysis or PFA), usually caused by one of three poliovirus serotypes (PV). The PV is eliminated wild in Brazil since 1989, cu rrently restricted to only four countries (Nigeria, Afghanistan, Pakistan and India). However PFA outbreaks associated with PV source vaccine and recombinant Non - polio enteroviruses has been reported. The characterization of EVNP is extremely important to investigate the diversity of co - circulating viruses, and to correlate clinical symptoms with viral serotype involved, including the investigation of routes of transmission of enteroviruses, during outbreaks, besides contributing for epidemiological studies and the evolution of enteroviruses. We analyzed samples related to AFP, using RT - PCR and PCR in order to identify which species of human enteroviruses associated. Members of the species C were sequenced for identification of serotype. From a total of 190 samples, 79 were of type C, 78 type B, 32 type A and one sample was corresponding for A and C, were not found samples of type D. Among the samples of species C, 58 corresponded to PV.Os data show similarities with similar studies in Europe and Asia, coveri ng a little noticed aspect of the epidemiology of enteroviruses in Brazilian territory.