The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-24T15:20:53Z No. of bitstreams: 1 DIOCRECIANO MATIAS BERO.pdf: 1601651 bytes, checksum: 383c1a12bee6df697cbcafb8f19017c7 (MD5) / Made available in DSpace on 2013-09-24T15:20:53Z (GMT). No. of bitstreams: 1 DIOCRECIANO MATIAS BERO.pdf: 1601651 bytes, checksum: 383c1a12bee6df697cbcafb8f19017c7 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os enterovírus humanos (HEV) são espécies do gênero Enterovirus, família Picornaviridae. Existem cerca de 120 sorotipos de HEV que são divididos em quatro espécies, designadas de HEV-A a D. Estes agentes infectam anualmente, milhões de pessoas no mundo, resultando em uma grande variedade de quadros clínicos que vão desde infecções inaparentes à febres inespecíficas, resfriado comum, à doenças graves, tais como meningite e poliomielite paralítica. As crianças são mais susceptiveis à infecção. A transmissão ocorre tanto pela via entérica e por via respiratória. O vírus pode ser excretado nas fezes por várias semanas. Este estudo teve como objectivo isolar e identificar os sorotipos de HEVs circulantes, a partir de amostras de fezes de crianças menores de 15 anos de idade, com quadros compatíveis a infecção por esses agentes, no Hospital Geral de Mavalane na Cidade de Maputo, em Moçambique. Neste trabalho, foram utilizadas 178 amostras de fezes obtidas entre novembro de 2011 a fevereiro de 2012. As amostras foram inoculadas em culturas de células e os enterovírus isolados foram identificados através de metódos moleculares, nas amostras negativas foi pesquisado o adenovírus. Das 45 amostras positivas em cultivos celulares, os enterovírus foram isolados e identificados em 26 (14,6 %). A proporção sexo masculino e feminino foi de 1,8: 1. O isolamento dos enterovírus diminuiu à medida que a idade aumentou. O sequenciamento gênomico revelou uma grande diversidade de enterovírus humanos. Entre os 26 enterovírus isolados, o Echovírus 29 foi o agente mais identificado com 19,2 %, seguido pelo Enterovírus 99 (11,5%). Foram identificados também Coxsackievírus A5, Echovírus sorotipos 11, 13 e Enterovírus C com 7,7 % de cada ; Coxsackievírus sorotipos A10, A13, A20, B4 e B6 com 3,85 % cada; Echovírus sorotipos 7, 21 e 25, com 3,85 % cada um, e Poliovírus sorotipos 2 e 3 com 3,85 %, respectivamente. Adenovírus foram isolados em 20 amostras, representando 11,2 % do total (20/178). Duas amostras apresentaram co-infecção enterovírus/adenovírus. Os resultados deste trabalho evidenciam a circulação de uma grande diversidade enterovírus humanos na cidade de Maputo, sendo os echovírus mais frequentes, mas também mostra a circulação de adenovírus humanos. Outros testes laboratoriais seriam necessários, para se relacionar inequivocamente a participação desses agentes virais na etiologia dos quadros clínicos observados. / The human enteroviruses (HEV) are species of the genus Enterovirus, family Picornaviridae. There are about 120 serotypes of HEV divided into four species, designated HEV- A to D. These agents infect millions of people worldwide each year, resulting in a wide variety of clinical conditions ranging from unapparent infection, undifferentiated fevers, and common cold to serious diseases such as meningitis and paralytic poliomyelitis. Children are more susceptible to infection. Transmission occurs by the fecal-oral and respiratory tract. The virus can be excreted in the feces for several weeks. The aim of this study was to isolate and identify human enteroviruses from stool samples of children less than 15 years of age presenting enterovirus compatible symptoms in Mavalane General Hospital in Maputo City, Mozambique. In this study, we used 178 stool samples from children under 15 years of age, obtained from November, 2011 to February, 2012. Samples were inoculated onto cell culture and the enterovirus isolates were identified by molecular methods, the negative samples was screened adenovirus. Twenty-six out of the 45 cell-culture positive samples were constituted by enteroviruses (14.6 %). The ratio between male and female was 1.8:1. Isolation of enterovirus decreases as the age increased. The genomic sequencing showed a diversity of human entrovirus. Among the 26 isolates, Echovirus serotype 29 was the most identified with 19.2 %; Coxsackievirus 99 was identified in 11.5 %, while Coxsackievirus A5, Echovirus serotypes 11, 13 and Enterovirus C, were identified in 7.7 % each. Coxsackievirus serotypes A10, A13, A20, B4 and B6 were present in 3.85 % each; Echovirus serotypes 7, 21 and 25, at 3.85 %, and poliovirus serotypes 2 and 3 in 3.85 %, respectively. Adenoviruses were isolated from 20 samples, 11.2 % (20/178). Two samples showed were co-infected with both enterovirus and adenoviruses. The results of this study showed a great diversity of enterovirus serotypes in the city of Maputo and echovirus was the most prevalent enterovirus found. We also showed the circulation of adenoviruses. However other laboratorial tests would be necessary in order to unequivocally correlate the participation of these viral agents in the etiology of the observed clinical findings.

Made available in DSpace on 2014-10-07T19:33:49Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 151.pdf: 1034631 bytes, checksum: 4633720fcfb8cd5c7e25a06a27819fe0 (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Rio de Janeiro, RJ, Brasil. / Infecções virais agudas do Sistema Nervoso Central (SNC), como as meningites e encefalites, são responsáveis por uma elevada morbidade, especialmente em crianças. Os enterovírus não-polio (EVNP) são responsáveis por mais de 80 por cento dos casos de meningite viral em que o agente etiológico é identificado. No presente estudo nós mostramos a freqüência de ocorrência de enterovírus em surtos e casos esporádicos de meningite viral no Brasil no período de dezembro de 1998 a dezembro de 2003. Das 1022 amostras de Líquido Cefalorraquidiano (LCR) analisadas pelo Laboratório de Enterovírus -FIOCRUZ, 162 (162/1022 -15,85 por cento) apresentaram isolamento viral em culturas de células RD e/ou HEp2. Destas, 139 (139/163 -85,27 por cento) foram identificadas por sequenciamento genômico parcial como echovírus 30. Outros enterovírus identificados foram: coxsackievírus B5 (6/162- 3,70 por cento), echovírus 13 (6/162- 3,70 por cento), echovírus 18 (5/162- 3,08 por cento), echovírus 6 (2/162- 1,23 por cento), echovírus 25 (2/162- 1,23 por cento), echovírus 1 (1/162- 0,61 por cento) e echovírus 4 (1/162- 0,61 por cento). A idade dos pacientes variou de 28 dias de vida a 68 anos. Os sintomas mais frequentes foram febre (77,00 por cento), cefaléia (69,51 por cento), ânsia de vômito (71,30 por cento), rigidez na nuca (41,35 por cento), convulsão (7,13 por cento) e diarréia (3,74 por cento). Durante o período de estudo, cinco surtos de meningite viral foram identificados e confirmados, sendo três no estado do Paraná (sul do Brasil), um em Pemambuco (nordeste do Brasil) e um no Rio Grande do Sul (sul do Brasil). Echovirus 30 causou quatro dos cinco surtos, e echovírus 13 foi responsável pelo último surto. Além dos surtos, 734 casos esporádicos também foram identificados durante o período de estudo e 59 destes casos apresentaram isolamento viral (59/734 -8,03 por cento). Echovírus 30 foi responsável pela maioria destes casos esporádicos (42/59 -71,18 por cento). Este estudo chama a atenção para o echovírus 30 como o principal agente etiológico envolvido em casos esporádicos e em surtos de meningite viral ocorridos no Brasil durante o período de estudo. / Acute viral infections of the Central Nervous System (CNS), as meningitis and encephalitis, are responsible for a high morbidity, particularly in children. Non-polio enteroviruses (NPEV) are responsible for over 80% of viral meningitis in which the etiologic agent is identified. In the present study we show the frequency of occurrence of enteroviruses in both outbreaks and sporadic cases of viral meningitis occurred in Brazil during the period of December 1998 to December 2003. From 1022 samples of Cerebrospinal Fluid (CSF) analyzed at the Enterovirus Laboratory, FIOCRUZ, 162 162/1022 - 15,85%) were positive regarding viral isolation in RD and/or HEp2 cells. From this total, 139 (139/162 - 85,27%) were identified by partial genome sequencing as being echovirus 30. Other identified enteroviruses were: coxsackievirus B5 (6/162 – 3,70%), echovirus 13 (6/162 - 3,70%), echovirus 18 (5/162 - 3,08%), echovirus 6 (2/162 - 1,23%), echovirus 25 (2/162 - 1,23%), echovirus 1 (1/162 - 0,61%) and echovirus 4 (1/162 0,61%). The age of the patients ranged from 28 days to 68 years old. The most frequent symptoms were fever (77,00%), headache (69,51%), vomiting (71,30%), neck stiffness 41,35%), convulsion (7,13%) and diarrhea (3,74%). Throughout the surveillance period, ive viral meningitis outbreaks were identified and confirmed, being three at Paraná State southern Brazil), one at Pernambuco (northeast Brazil) and one at Rio Grande do Sul southern Brazil). Echovirus 30 caused four out of the five outbreaks, and echovirus 13 caused the last one. Besides the outbreaks, 734 sporadic clinical cases were also identified during the period of study and 59 of these were positive with regard viral isolation (59/734 8,03%). Echovirus 30 accounted for the majority of these sporadic cases (42/59 – 71,18%). This study calls the attention to the echovirus 30 as the most prevalent etiological agent involved in both sporadic cases and outbreaks of viral meningitis occurred in Brazil during the period of study.

Made available in DSpace on 2014-07-28T18:10:50Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 97.pdf: 1747812 bytes, checksum: ac9891f962a8dc55e948eb9245ec6dc5 (MD5) Previous issue date: 2008 / Made available in DSpace on 2014-12-22T16:56:35Z (GMT). No. of bitstreams: 2 97.pdf: 1747812 bytes, checksum: ac9891f962a8dc55e948eb9245ec6dc5 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Os enterovírus estão entre os mais comuns vírus humanos e são de grande interesse devido à ampla variedade de infecções que podem causar. A vigilância das Paralisias Flácidas Agudas (PFAs) e o diagnóstico laboratorial dos enterovírus são partes críticas da iniciativa da Organização Mundial de Saúde para erradicação mundial da poliomielite, assim como a necessidade de disponibilizar técnicas rápida para o diagnóstico diferencial destes vírus. A caracterização dos enterovírus é importante para a investigação da diversidade de vírus co-circulantes, para determinar a correlação entre dados celulares e bioquímicos durante a infecção,para relacionar o tipo de sintoma clínico com o sorotipo enteroviral, incluindo a investigação de vias de transmissão de enterovírus durante épocas de surtos. Além disso, a caracterização dos enterovírus é de extrema relevância para distinguir as infecções provocadas pelos Poliovirus dos enterovírus não-pólio no contexto do Programa de Erradicação da Poliomielite da OMS. O presente estudo teve como objetivo principal identificar, através da técnica de RT-PCR e seqüenciamento nucleotídico, a presença de enterovírus diretamente das amostras de primeira passagem de cultura celular a fim de diminuir o custo e o tempo de liberação do diagnóstico. Para isso, foram analisadas 221 amostras de casos suspeitos de PFA, inoculadas em primeira passagem de cultura de células RD. Destas, 17 foram positivas para enterovírus. A comparação da técnica com a indicada pela OMS mostrou alta sensibilidade e especificidade, indicando que a nova metodologia pode ser seguramente empregada como forma de garantia de um diagnóstico mais rápido. / The enterovirus are among the most common human viruses, and are of great interest because of the wide variety of infections that can cause. The surveillance of Acute Flaccid Paralysis (AFP) and laboratory diagnosis of enterovirus are critical parts of the initiative of the World Health Organization (WHO) to eradicate polio worldwide, as well as the availability of rapid completion of techniques are needed for the differential diagnosis of these viruses. The characterization of enterovirus is important for the research of the diversity of virus co-circulating, to determine the correlation between cellular and biochemical data during infection, relate to the type of clinical symptoms with the serotype enteroviral, including investigation of routes of transmission of enterovirus during times of outbreaks. Moreover, the characterization of enterovirus is of extreme importance to distinguish Poliovirus infections caused by the enterovirus non pólio in the context of the Program for the Eradication of Poliomyelitis of WHO. The aim of this study is to identify, through RT-PCR and nucleotide sequencing, the presence of enterovírus genome directly from first passage of cell culture in order to reduce the cost and time of release of diagnosis. For that, were analyzed 221 samples of suspected cases of FAP, inoculated in first passage of RD cell culture. Seventeen samples were positive for enterovirus. The comparison this technique with the indicated by the WHO showed high sensitivity and specificity, indicating that the new method can be safely employed as a way of ensuring a faster diagnosis.

Les Entérovirus humains du groupe B (EV-B) et plus spécifiquement les virus Coxsackie B sont considérés comme une cause majeure des myocardites infectieuses aigues et chroniques dont 10% peuvent évoluer vers la cardiomyopathie dilatée (CMD). Les mécanismes moléculaires viraux impliqués dans la progression de la myocardite aiguë vers le stade de la CMD ne sont pas élucidés.L’analyse par séquençage NGS a montré chez 8 (33%) des 24 patients atteints de CMD inexpliquée l’existence de populations majoritaires tronquées de 19 à 50 nucléotides associées à des formes virales minoritaires complètes. La proportion de populations tronquées s’est révélée négativement corrélée au ratio ARN+/ARN- et à la charge virale. Des études immuno-histologiques et par hybridation in situ des tissus cardiaques ont montré que le clivage de la dystrophine était uniquement retrouvé dans les cardiomyocytes infectés par les EV-B. Pour étudier les activités de réplication des populations d’EV-B persistants, un réplicon (CVB3-emGFP) a été généré à partir d’une souche cardiotrope (CV-B3/28). La transfection d’ARN de synthèse complets et tronqués (d50) dans des cultures de cardiomyocytes humains primaires a mis en évidence des mécanismes de recombinaison et/ou de trans-complémentation entre ces 2 formes virales induisant de faibles activités de réplication.Nos résultats démontrent l’existence de mécanismes de coopération moléculaire entre des populations d’EV-B tronquées et complètes qui pourraient expliquer la mise en place du mécanisme de persistance virale observée au cours de la phase clinique de CMD. Ces résultats pourraient contribuer au développement de nouvelles stratégies thérapeutiques pour prévenir et traiter les infections cardiaques à EV-B. / Enteroviruses group B (EV-B) and more specifically Coxsackievirus B are recognized as major causes of acute and chronic infectious myocarditis, which 10% may progress towards dilated cardiomyopathy (DCM). Viral molecular mechanisms involved in the progression from acute myocarditis to the clinical stage of DCM remain unknown.Deep sequencing analysis showed in 8 (33%) of 24 unexplained DCM patients the existence of major CVB3 populations with deletions of 19 to 50 nucleotides associated with a minority of complete viral forms. The proportion of deleted viral populations was negatively correlated with RNA+/RNA- ratio and the viral load levels. Immuno-histological and in situ hybridization assays of DCM cardiac tissues demonstrated that the cleavage of dystrophin was found only in cardiomyocytes infected with EV-B. To study the replication activities of persistent EV-B populations, a replicon (CVB3-emGFP) was generated from a cardiotropic strain (CV-B3/28). Transfection of synthesized complete and truncated (d50) viral RNAs in primary human cardiomyocytes cultures revealed mechanisms of recombination and / or trans-complementation between these two viral forms inducing low replication activities.In conclusions, our original results demonstrated the existence of new molecular mechanisms of cooperation between EV-B deleted and complete viral populations that could explain the development of a viral persistence mechanism observed during the clinical phase of DCM. These findings may contribute to the development of new therapeutic strategies to prevent and treat persistent heart EV-B infections.

La présente étude vise à étudier le potentiel de bifidobactéries à protéger les cellules contre l’infection par le Coxsackie B4 (CV-B4). Le criblage de bifidobactéries a identifié deux des cinq souches qui protégeaient les cellules HEp-2 lorsque les bifidobactéries sont pré-incubées avec les particules virales avant l'inoculation sur les cellules Hep-2. En revanche, aucun effet protecteur n'a été observé en incubant les cellules Hep-2 avec des bifidobactéries avant l'inoculation de CV-B4. Les lipoprotéines des parois cellulaires (LpAs) sécrétées par les souches sélectionnées sont testées pour leur activité antivirale. Les deux LpAs présentaient une activité antivirale quand ils sont incubés avec les particules virales avant d’être inoculées aux cellules HEp-2. Aucun effet protecteur n'a été induit par incubation des LpAs avec les cellules HEp-2 avant l'inoculation de CV-B4. La protéine recombinante présente une activité antivirale identique. Pour identifier les séquences peptidiques interagissant avec les particules virales, les protéines de LpAs sont alignées avec les séquences peptidiques du nord bord du canyon et avec l’empreinte de la région puff sur le Coxsackievirus et sur le récepteur de l’adénovirus (CAR). L'étude d'amarrage moléculaire in silico (Docking) utilisant le CV-B3 en tant que modèle a montré une faible énergie de liaison indiquant un système stable pour les peptides sélectionnés et par conséquent une interaction probable avec le CV-B. Les peptides de B.longum et de B.breve qui sont homologues à l’empreinte du rebord nord viral sur la séquence CAR, forment des liaisons d’hydrogène avec plusieurs résidus viraux dans la région du rebord nord du canyon, qui sont déjà décrits pour leur interaction avec le CAR.En conclusion, les protéines de LPAS bifidobactéries peuvent inhiber l'infection par le CV-B4 probablement par liaison aux acides aminés de la capside qui interagissent avec le CAR. / The present study aims at investigating the potential of bifidobacteria in protecting cells from Coxsackievirus B4 (CV-B4) infection. The bifidobacterial screening identified two out of five strains that protected HEp-2 cell viability when bifidobacteria were incubated with the viral particles prior inoculation. In contrast, no effect was shown by incubating HEp-2 cells with bifidobacteria prior CV-B4 inoculation. Cell-wall lipoproteins secreted by the selected strains (LpAs) were assayed for their anti-viral activity. The two LpAs exhibited anti-viral activity when they were incubated with the viral particles prior inoculation to HEp-2 cells. No effect was induced by incubating LpAs with HEp-2 cells prior CV-B4 inoculation. The recombinant LpAs derived-protein exhibited identical anti-viral activity. To identify the peptide sequences interacting with the virus particles, LpAs proteins were aligned with the peptide sequences of north canyon rim and puff footprint onto coxsackievirus and adenovirus receptor (CAR). The in silico molecular docking study using CV-B3 as template showed a low energy binding indicating a stable system for the selected peptides and consequently a likely binding interaction with CV-B. B.longum and B.breve peptides homologous to the viral north rim footprint onto CAR sequence formed hydrogen bonds with several viral residues in the north rim of the canyon, which were already predicted as interacting with CAR. In conclusion, proteins from bifidobacterial LpAs can inhibit the infection with CV-B4 likely through binding to the capsid aminoacids that interact with CAR.

Parce qu’ils sont la première cause virale d’infections des voies respiratoires supérieures et inférieures, les rhinovirus humains (RVH) constituent un problème majeur de santé publique. À ce jour, aucun vaccin ni antiviral n’est disponible pour lutter contre ces agents pathogènes. Un crible en doublehybride chez la levure nous a permis d’identifier un nouveau partenaire peptidique de la protéase virale à cystéine 2A (2Apro), l’hexapeptide LVLQTM. Ce dernier agit comme un véritable pseudosubstrat de la 2Apro et inhibe son activité. Ce peptide a été modifié chimiquement à son extrémité C-terminale avec un groupement réactif électrophile fluorométhylcétone pour former une liaison covalente avec le groupement thiol nucléophile du site actif de l'enzyme viral. Des expériences réalisées ex vivo et in vivo ont montré que le peptide LVLQTM modifié était un puissant inhibiteur de la réplication du RVH dans les cellules A549 et chez la souris. La structure 3D déjà connue de la 2Apro du RVH-2 a ensuite permis de modéliser la fixation de LVLQTM dans la poche de liaison du substrat de la protéase et la comparaison des séquences des 2Apro des espèces RVH-A, -B et -C a révélé que les résidus impliqués dans l'interaction avec le peptide LVLQTM sont relativement bien conservés. Si le peptide inhibiteur semblait donc agir contre tous les sérotypes de RVH, son utilisation à des fins thérapeutiques pouvait être étendue à d'autres entérovirus puisqu’il inhibait également la 2Apro de l’entérovirus 71 (EV-71) et par conséquent la réplication virale. De plus, la comparaison de la séquence des protéases 2A de l’EV-71 avec celle du RVH-A2 n’a révélé aucune différence majeure. Par conséquent, cette étude ouvre de nouvelles perspectives dans la mise au point d’un antiviral à large spectre d’action contre tous les entérovirus / Human rhinoviruses (HRV) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. To date no vaccine or antiviral are available against these pathogens. Using a high-throughput yeast two-hybrid screening, we identified a six amino acid “hit” peptide, LVLQTM, which acted as a pseudo-substrate of the viral 2A cysteine protease (2Apro) and inhibited its activity. This peptide was chemically modified at its C-terminus with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. Based on HRV-2 2Apro crystallographic data, a virtual docking model was then set up to predict the inhibitor binding mode into the ligand binding pocket of the enzyme. Sequence comparison between different 2Apro from HRV-A, -B and –C species revealed that the aminoacid residues involved in the interaction with the inhibitor are relatively well conserved. If our peptide inhibitor seemed to be of general use against all HRV serotypes, its use for therapeutic purposes could be extended to other enterovirus-associated diseases since it was also active against Human Enterovirus 71 (EV-71) 2A proteases and EV-71 replication. Moreover, comparison of the sequence of these proteases with the one of HRV-A2 revealed only minor differences in the residues involved in the interaction with LVLQTM. Therefore, this study opens new doors in the development of an antiviral against a wide range of enteroviruses

<p><i>Enterovirus</i> (EV), a genus within the <i>Picornaviridae </i>family, contains icosahedral positive-stranded RNA viruses linked to different human and mammalian diseases with a variety of symptoms ranging from the common cold to central nervous system infection. An important member within this genus is EV-D68. Unlike many enteroviruses that use the gastrointestinal tract as the transmission and propagation route, EV-D68 infects the respiratory tract and causes respiratory illness, especially in children. Severe infections of EV-D68 also lead to acute flaccid myelitis (AFM), a polio-like neurological disease. Especially in recent years, EV-D68 has been on a global upswing. However, no antiviral interventions against EV-D68 infection have been developed to date. Antibodies neutralizing EV-D68 have significant vaccine and therapeutic potentials. Here, the structures of the immune complex between EV-D68 and the Fab molecules of EV-D68 human monoclonal antibodies have been reconstructed using cryo-electron microscopy (cryo-EM). These structures show two Fab binding loci on the virion surface as well as the essential amino acids involved in binding. In addition to antibodies, a drug candidate against EV-D68 has been investigated in this work as an antiviral strategy. It is likely that this drug blocks viral entry through binding in the hydrophobic pocket underneath the viral protein 1, the largest structural protein of EV-D68. Furthermore, the morphogenesis of EV-D94, another causative virus of polio-like disease, which is closely related to EV-D68 with 85% sequence identity, has been investigated using cryo-EM. Compared to EV-D68, the shape of the canyon and the loops containing the immunogenic recognition sites are different in EV-D94. The structures of each of the three stages of EV-D94 particles (the full native virion, the uncoating intermediate, and the empty virion) were identified and delineate the viral uncoating process. These findings reveal useful knowledge and new insights to develop treatments against human EVs. </p><p></p>