The Interferon (IFN) family of cytokines are produced in direct response to virus infection and they constitute the first line of defence against virus infection by inducing hundreds of interferon stimulated genes (ISGs) which act in concert to establish the so-called “antiviral state”. Influenza A viruses and parainfluenza virus type 5 (PIV5) are both small negative strand RNA viruses that must circumvent their hosts’ interferon (IFN) response for replication. However, the ways in which these viruses interact with the IFN system are very different. Although PIV5 replication is initially severely impaired in cells in a pre-existing IFN-induced antiviral state, it manages to overcome the antiviral state by targeting an essential component of type I IFN signalling, STAT1, for degradation. Thus the cells cannot maintain the antiviral state indefinitely without continuous signalling. Consequently, the virus resumes its normal replication pattern after 24-48 hours post-infection. In clear contrast, influenza virus fails to establish its replication in the majority of infected cells (90-95%) with a pre-existing IFN-induced antiviral state, although a few cells are still able to produce viral antigens. To further investigate how influenza virus interacts with cells in a pre-existing IFN-induced antiviral state, I have used in situ hybridization to follow the fate of input and progeny genomes in cells that have, or have not, been treated with IFN prior to infection. Here I show for the first time that IFN pre-treatment blocks the nuclear import of influenza A virus genome, which prevents the establishment of virus replication, but this can be overcome by increasing multiplicities of infection. Of those IFN-induced antiviral molecules, human MxA is an essential component of the IFN-induced antiviral state in blocking influenza virus genome import, as this block can be abolished by lentivirus-mediated knockdown of MxA. I also show that in cells constitutively expressing MxA the viral genome still manages to be transported into the nucleus, indicating that MxA might require an unidentified IFN-induced factor to block nuclear import of the influenza virus genome. These results reveal that IFN exerts its action at an early stage of virus infection by inducing MxA to interfere with the transport of viral genome into the nucleus, which is the factory for viral RNA production.

Bei bhPIV3 handelt es sich um ein bovines Parainfluenzavirus Typ 3 (bPIV3), dessen Ober-flächenproteingene gegen jene des humanen Parainfluenzavirus Typ 3 (hPIV3) ausgetauscht wurden. Dieses ursprünglich als experimenteller Impfstoff gegen hPIV3 entwickelte Virus wurde darüber hinaus als Impfvektor zur Expression anderer viraler Antigene verwendet. Im Rahmen der hier vorgestellten Arbeit wurden die ersten bhPIV3-basierten Vektoren für nicht-virale Antigene hergestellt und in einem ersten Versuch evaluiert. Dazu wurden ein reverses Genetiksystem zur Herstellung rekombinanter bhPIV3 in einem neuen Labor aufgebaut und fünf neue rekombinante Viren erhalten, welche zusätzlich Antigene des Mycobacterium tuberculosis (M. tb.) exprimieren. Balb/c-Mäuse wurden intranasal mit den bhPIV3-Vektoren infiziert, so dass sowohl deren Replikation als auch der induzierte protektive Effekt gegenüber M. tb.-Neuinfektionen getestet werden konnte. In einem ersten Versuch zeigte sich, dass eine Immunisierung mit den rekombinanten Viren allein keine Schutzwirkung entfaltet. Als Boost-Impfung nach Gabe des Bacille Calmette Guérin (BCG) zeigten einige Vektoren jedoch einen signifikanten protektiven Effekt. In einem Folgeversuch konnten diese Beobachtungen jedoch bislang nicht bestätigt werden, so dass weitere Versuche durchzuführen sind, bevor eine endgültige Aussage bezüglich des hervorgerufenen Schutzeffektes getroffen werden kann. In einem weiteren Tierversuch wurde gezeigt, dass die Baumwollratte ein Tiermodell darstellt, in dem bhPIV3 erheblich schlechter repliziert als hPIV3. Trotz der eingeschränkten Replikation induzierte bhPIV3 neutralisierende Antikörpertiter gegen hPIV3, die mit durch hPIV3 induzierten Titern vergleichbar waren. Mit Hilfe eines neu generierten rekombinanten Virus, welches das grün fluoreszierende Protein EGFP exprimiert, konnte ein Weg aufgewiesen werden, die Bestimmung neutralisierender Antikörpertiter deutlich zu vereinfachen. / The initial objective of this project was to establish a reverse genetic system for generation of recombinant bovine/human parainfluenza virus type 3 (bhPIV3), a bovine PIV3 (bPIV3) in which the bhPIV3 glycoprotein genes are replaced by their counterparts of human PIV3 (hPIV3). In addition, methods needed to characterise virus infectivity, genetic integrity and relevant in vitro phenotypes were established. The reverse genetics system was used to add individual mycobacterium tuberculosis (M. tb.) open reading frames (ORFs) as supernumerary gene units to the bhPIV3 genome and to rescue bhPIV3 vectors that expressed M. tb. antigens. In addition, a similar vector expressing the enhanced green fluorescent protein (EGFP) was constructed. Following the in vitro characterization of the derived viral vectors, the M. tb. vectors were evaluated for their efficacy to protect against M. tb. aerosole challenge in the Balb/c mouse model for tuberculosis. Although, in a single experiment, vaccination with bhPIV3 vectors alone did not confer any protection against M. tb. challenge, a boost with selected bhPIV3 vectors after Bacille Calmette Guérin (BCG) priming was successful in conferring protective efficacy against M. tb. challenge. A repeat of this challenge study could not confirm the initial observation, and further experiments are needed to determine whether the observed protection can be reliably reproduced. Evaluation of the bhPIV3 vectors in the cotton rat model showed that this small animal model is suitable to evaluate the attenuation phenotype of bhPIV3 compared to human parainfluenza virus type 3 (hPIV3). Although replication of bhPIV3 was highly restricted compared to hPIV3, hPIV3 neutralizing antibody titers induced by bhPIV3 infection were similar to those induced by hPIV3 infection. Studies with bhPIV3 expressing EGFP led to a new fluorescence based assay to determine hPIV3 neutralizing antibody titers. This assay could save time and resources in hPIV3 serology.

O vírus da cinomose canina (CDV) e o vírus parainfluenza canino (CPIV) afetam uma ampla variedade de hospedeiros e encontram-se distribuídos mundialmente. O CDV é considerado um dos mais importantes agentes infecciosos dentro das populações caninas. Este vírus é o agente causal da cinomose, uma doença potencialmente letal em membros das famílias Canidae, Mustelidae e Procionidae, sendo recentemente detectado como causa de morbidade e mortalidade em carnívoros aquáticos e grandes felinos. O CPIV, por sua vez, é altamente contagioso entre cães, podendo infectar roedores e gatos em infecções experimentais. Geralmente, o CPIV produz uma traqueobronquite aguda auto-limitante, porém pode atuar sinergicamente com outros agentes infecciosos, como o CDV, causando sinais clínicos mais graves. Como em nosso meio são escassas as informações sobre estes vírus, o presente estudo visou aprofundar os conhecimentos sobre a prevalência de CDV e CPIV em cães e felinos silvestres mantidos em cativeiro. Para tanto, soros destes animais foram testados em busca de anticorpos neutralizantes contra amostras padrão do CDV (Rockborn e Snyder Hill) e do CPIV (V660). Inicialmente, foram testados soros de 173 cães de rua mantidos em canis municipais em Novo Hamburgo e Porto Alegre, RS. A prevalência de anticorpos neutralizantes anti-CDV frente às amostras de vírus da cinomose Rockborn e Snyder Hill, foi de 9,3 % e 4,1 %, respectivamente. Somente dois cães apresentaram títulos de anticorpos considerados protetores contra CDV Rockborn (igual ou maior que 100) e nenhum soro apresentou título de anticorpos neutralizantes considerado protetor para a amostra Snyder Hill (igual ou maior que 100). Contra a amostra de parainfluenza canino V660, a prevalência de anticorpos neutralizantes encontrada foi de 51,4 %. Conclui-se, portanto, que a população de cães de rua amostrada apresenta poucos indícios de contato prévio com CDV, sugerindo grande susceptibilidade à cinomose. Por outro lado, o CPIV parece circular amplamente nesta população. Na segunda parte do presente estudo, como no Brasil não existem relatos sobre CDV e CPIV em felinos silvestres, buscou-se verificar a possibilidade da ocorrência dessas infecções em felinos silvestres brasileiros. Para tanto, foram testados soros de 84 felinos silvestres de seis diferentes espécies nativas do Brasil (Leopardus tigrinus, Puma concolor, Leopardus wiedii, Herpailurus yaguarondi, Panthera onca), todos mantidos em cativeiro em criatórios de distintas regiões do País. Todos os felinos amostrados apresentaram-se soronegativos frente às amostras de CDV e CPIV utilizadas. Estes resultados indicam que CDV e CPIV parecem não circular nas populações de felinos silvestres amostradas. / Canine distemper virus (CDV) and canine parainfluenza virus (CPIV) infect a great variety of hosts ranges and are distributed worldwide. CDV is one of the most important infectious agents in dogs. This virus may cause potentially lethal disease among members of the Canidae, Mustelidae and Procionidae families. It has also caused diseases of significant morbidity and mortality in aquatic carnivores and large felids. CPIV, on its turn, is highly contagious among dogs, whilst rodents and cats are susceptible to experimental infections. CPIV is usually associated with an acute selflimiting tracheobronchitis. However, it can act sinergistically with other infectious agents, such as CDV, and cause clinical signs of variable severity. As information on CDV and CPIV infections in our millieu are scarce, this study was carried out aiming to increase knowledge on the prevalence of CDV and CPIV in stray dogs as well as in captive Brazilian wild felids. In order to have an estimate on such prevalences, sera from these animals were tested for neutralizing antibodies to CDV strains Rockborn and Snyder Hill, and to CPIV strain V660. Initially, 173 sera from stray dogs kept in kennels from the municipalities of Novo Hamburgo and Porto Alegre, RS, were examined. The prevalences of neutralizing antibodies to CDV strains Rockborn and Snyder Hill were 9.3 % and 4.1 %, respectively. Only two dogs had antibody levels which could be correlated to protection (that is, titre ≥ 100) to CDV Rockborn whereas no sera presented antibody titres high enough to be considered protective to CDV strain Snyder Hill (that is, titre ≥ 100). Regarding CPIV, the prevalence of anti-V660 neutralizing antibodies was 51.4 %. It can be concluded that the stray dog populations under study shows few serological evidence of previous contact with CDV and seem largely susceptible to CDV infections. On the other hand, CPIV seems to circulate widely in the examined population. In the second part of this study, as there are no reports on CDV and CPIV infections in wild felids in Brazil, it was aimed to determine whether there would be any evidence of such infections among some of such species. For that, 84 sera from wild felids of six different Brazilian native species (Leopardus tigrinus, Puma concolor, Leopardus wiedii, Herpailurus yaguarondi, Panthera onca), all kept in captivity in different regions of the country, were tested for neutralizing antibodies to both CDV and CPIV. All wild felid sera tested were negative for antibodies to the two strains of CDV as well as to CPIV. These results indicate that CDV and CPIV do not seem to circulate among the wild felid populations examined.

O vírus da cinomose canina (CDV) e o vírus parainfluenza canino (CPIV) afetam uma ampla variedade de hospedeiros e encontram-se distribuídos mundialmente. O CDV é considerado um dos mais importantes agentes infecciosos dentro das populações caninas. Este vírus é o agente causal da cinomose, uma doença potencialmente letal em membros das famílias Canidae, Mustelidae e Procionidae, sendo recentemente detectado como causa de morbidade e mortalidade em carnívoros aquáticos e grandes felinos. O CPIV, por sua vez, é altamente contagioso entre cães, podendo infectar roedores e gatos em infecções experimentais. Geralmente, o CPIV produz uma traqueobronquite aguda auto-limitante, porém pode atuar sinergicamente com outros agentes infecciosos, como o CDV, causando sinais clínicos mais graves. Como em nosso meio são escassas as informações sobre estes vírus, o presente estudo visou aprofundar os conhecimentos sobre a prevalência de CDV e CPIV em cães e felinos silvestres mantidos em cativeiro. Para tanto, soros destes animais foram testados em busca de anticorpos neutralizantes contra amostras padrão do CDV (Rockborn e Snyder Hill) e do CPIV (V660). Inicialmente, foram testados soros de 173 cães de rua mantidos em canis municipais em Novo Hamburgo e Porto Alegre, RS. A prevalência de anticorpos neutralizantes anti-CDV frente às amostras de vírus da cinomose Rockborn e Snyder Hill, foi de 9,3 % e 4,1 %, respectivamente. Somente dois cães apresentaram títulos de anticorpos considerados protetores contra CDV Rockborn (igual ou maior que 100) e nenhum soro apresentou título de anticorpos neutralizantes considerado protetor para a amostra Snyder Hill (igual ou maior que 100). Contra a amostra de parainfluenza canino V660, a prevalência de anticorpos neutralizantes encontrada foi de 51,4 %. Conclui-se, portanto, que a população de cães de rua amostrada apresenta poucos indícios de contato prévio com CDV, sugerindo grande susceptibilidade à cinomose. Por outro lado, o CPIV parece circular amplamente nesta população. Na segunda parte do presente estudo, como no Brasil não existem relatos sobre CDV e CPIV em felinos silvestres, buscou-se verificar a possibilidade da ocorrência dessas infecções em felinos silvestres brasileiros. Para tanto, foram testados soros de 84 felinos silvestres de seis diferentes espécies nativas do Brasil (Leopardus tigrinus, Puma concolor, Leopardus wiedii, Herpailurus yaguarondi, Panthera onca), todos mantidos em cativeiro em criatórios de distintas regiões do País. Todos os felinos amostrados apresentaram-se soronegativos frente às amostras de CDV e CPIV utilizadas. Estes resultados indicam que CDV e CPIV parecem não circular nas populações de felinos silvestres amostradas. / Canine distemper virus (CDV) and canine parainfluenza virus (CPIV) infect a great variety of hosts ranges and are distributed worldwide. CDV is one of the most important infectious agents in dogs. This virus may cause potentially lethal disease among members of the Canidae, Mustelidae and Procionidae families. It has also caused diseases of significant morbidity and mortality in aquatic carnivores and large felids. CPIV, on its turn, is highly contagious among dogs, whilst rodents and cats are susceptible to experimental infections. CPIV is usually associated with an acute selflimiting tracheobronchitis. However, it can act sinergistically with other infectious agents, such as CDV, and cause clinical signs of variable severity. As information on CDV and CPIV infections in our millieu are scarce, this study was carried out aiming to increase knowledge on the prevalence of CDV and CPIV in stray dogs as well as in captive Brazilian wild felids. In order to have an estimate on such prevalences, sera from these animals were tested for neutralizing antibodies to CDV strains Rockborn and Snyder Hill, and to CPIV strain V660. Initially, 173 sera from stray dogs kept in kennels from the municipalities of Novo Hamburgo and Porto Alegre, RS, were examined. The prevalences of neutralizing antibodies to CDV strains Rockborn and Snyder Hill were 9.3 % and 4.1 %, respectively. Only two dogs had antibody levels which could be correlated to protection (that is, titre ≥ 100) to CDV Rockborn whereas no sera presented antibody titres high enough to be considered protective to CDV strain Snyder Hill (that is, titre ≥ 100). Regarding CPIV, the prevalence of anti-V660 neutralizing antibodies was 51.4 %. It can be concluded that the stray dog populations under study shows few serological evidence of previous contact with CDV and seem largely susceptible to CDV infections. On the other hand, CPIV seems to circulate widely in the examined population. In the second part of this study, as there are no reports on CDV and CPIV infections in wild felids in Brazil, it was aimed to determine whether there would be any evidence of such infections among some of such species. For that, 84 sera from wild felids of six different Brazilian native species (Leopardus tigrinus, Puma concolor, Leopardus wiedii, Herpailurus yaguarondi, Panthera onca), all kept in captivity in different regions of the country, were tested for neutralizing antibodies to both CDV and CPIV. All wild felid sera tested were negative for antibodies to the two strains of CDV as well as to CPIV. These results indicate that CDV and CPIV do not seem to circulate among the wild felid populations examined.

O vírus da cinomose canina (CDV) e o vírus parainfluenza canino (CPIV) afetam uma ampla variedade de hospedeiros e encontram-se distribuídos mundialmente. O CDV é considerado um dos mais importantes agentes infecciosos dentro das populações caninas. Este vírus é o agente causal da cinomose, uma doença potencialmente letal em membros das famílias Canidae, Mustelidae e Procionidae, sendo recentemente detectado como causa de morbidade e mortalidade em carnívoros aquáticos e grandes felinos. O CPIV, por sua vez, é altamente contagioso entre cães, podendo infectar roedores e gatos em infecções experimentais. Geralmente, o CPIV produz uma traqueobronquite aguda auto-limitante, porém pode atuar sinergicamente com outros agentes infecciosos, como o CDV, causando sinais clínicos mais graves. Como em nosso meio são escassas as informações sobre estes vírus, o presente estudo visou aprofundar os conhecimentos sobre a prevalência de CDV e CPIV em cães e felinos silvestres mantidos em cativeiro. Para tanto, soros destes animais foram testados em busca de anticorpos neutralizantes contra amostras padrão do CDV (Rockborn e Snyder Hill) e do CPIV (V660). Inicialmente, foram testados soros de 173 cães de rua mantidos em canis municipais em Novo Hamburgo e Porto Alegre, RS. A prevalência de anticorpos neutralizantes anti-CDV frente às amostras de vírus da cinomose Rockborn e Snyder Hill, foi de 9,3 % e 4,1 %, respectivamente. Somente dois cães apresentaram títulos de anticorpos considerados protetores contra CDV Rockborn (igual ou maior que 100) e nenhum soro apresentou título de anticorpos neutralizantes considerado protetor para a amostra Snyder Hill (igual ou maior que 100). Contra a amostra de parainfluenza canino V660, a prevalência de anticorpos neutralizantes encontrada foi de 51,4 %. Conclui-se, portanto, que a população de cães de rua amostrada apresenta poucos indícios de contato prévio com CDV, sugerindo grande susceptibilidade à cinomose. Por outro lado, o CPIV parece circular amplamente nesta população. Na segunda parte do presente estudo, como no Brasil não existem relatos sobre CDV e CPIV em felinos silvestres, buscou-se verificar a possibilidade da ocorrência dessas infecções em felinos silvestres brasileiros. Para tanto, foram testados soros de 84 felinos silvestres de seis diferentes espécies nativas do Brasil (Leopardus tigrinus, Puma concolor, Leopardus wiedii, Herpailurus yaguarondi, Panthera onca), todos mantidos em cativeiro em criatórios de distintas regiões do País. Todos os felinos amostrados apresentaram-se soronegativos frente às amostras de CDV e CPIV utilizadas. Estes resultados indicam que CDV e CPIV parecem não circular nas populações de felinos silvestres amostradas. / Canine distemper virus (CDV) and canine parainfluenza virus (CPIV) infect a great variety of hosts ranges and are distributed worldwide. CDV is one of the most important infectious agents in dogs. This virus may cause potentially lethal disease among members of the Canidae, Mustelidae and Procionidae families. It has also caused diseases of significant morbidity and mortality in aquatic carnivores and large felids. CPIV, on its turn, is highly contagious among dogs, whilst rodents and cats are susceptible to experimental infections. CPIV is usually associated with an acute selflimiting tracheobronchitis. However, it can act sinergistically with other infectious agents, such as CDV, and cause clinical signs of variable severity. As information on CDV and CPIV infections in our millieu are scarce, this study was carried out aiming to increase knowledge on the prevalence of CDV and CPIV in stray dogs as well as in captive Brazilian wild felids. In order to have an estimate on such prevalences, sera from these animals were tested for neutralizing antibodies to CDV strains Rockborn and Snyder Hill, and to CPIV strain V660. Initially, 173 sera from stray dogs kept in kennels from the municipalities of Novo Hamburgo and Porto Alegre, RS, were examined. The prevalences of neutralizing antibodies to CDV strains Rockborn and Snyder Hill were 9.3 % and 4.1 %, respectively. Only two dogs had antibody levels which could be correlated to protection (that is, titre ≥ 100) to CDV Rockborn whereas no sera presented antibody titres high enough to be considered protective to CDV strain Snyder Hill (that is, titre ≥ 100). Regarding CPIV, the prevalence of anti-V660 neutralizing antibodies was 51.4 %. It can be concluded that the stray dog populations under study shows few serological evidence of previous contact with CDV and seem largely susceptible to CDV infections. On the other hand, CPIV seems to circulate widely in the examined population. In the second part of this study, as there are no reports on CDV and CPIV infections in wild felids in Brazil, it was aimed to determine whether there would be any evidence of such infections among some of such species. For that, 84 sera from wild felids of six different Brazilian native species (Leopardus tigrinus, Puma concolor, Leopardus wiedii, Herpailurus yaguarondi, Panthera onca), all kept in captivity in different regions of the country, were tested for neutralizing antibodies to both CDV and CPIV. All wild felid sera tested were negative for antibodies to the two strains of CDV as well as to CPIV. These results indicate that CDV and CPIV do not seem to circulate among the wild felid populations examined.

Master of Veterinary Biomedical Sciences / Department of Clinical Sciences / Bradley White / Objective- To compare serologic response and viral replication following intranasal administration of a modified-live bovine rhinotracheitis (IBR) parainfluenza-3 (PI-3) vaccine in high (32°C) and moderate (21°C) ambient temperatures. Animals- 28 heifers (mean body weight, 206.8 kg) Procedures- Heifers randomly allocated to treatment groups: High Ambient Temperature (HAT, n=10): received vaccine, housed outdoors, Moderate Ambient Temperature (MAT, n=10): received vaccine, housed indoors, High Ambient Control (HAC, n=4): no vaccine, housed outdoors, Moderate Ambient Control (MAC, n=4): no vaccine, housed indoors. Rectal and nasal mucosal temperatures were recorded every 2 hours from 8am to 8pm on trial days 0 and 1. Nasal swabs were collected on trial days 0 through 7 for virus isolation. Serum samples were collected for serology on trial days 0, 7, 14, and 28. Results- Rectal temperatures did not differ among treatment groups over the study period, but nasal temperatures were higher in the HAT calves compared to MAT group at study hours: 6, 24, 30, 32, and 38. Two weeks post-vaccination, IBR titers were significantly greater in vaccinates (HAT,MAT) relative to non-vaccinates (HAC, LAC), but no differences were identified among HAT and MAT. Viable IBR virus was recovered via virus isolation from all vaccinated calves (HAT,MAT) on trial days 1 through 6. Conclusions and Clinical Relevance- The ability to isolate IBR and stimulate the calf immune response following administration of a modified-live IBR-PI3 intranasal vaccine did not differ in calves housed in temperature-controlled and high ambient temperature environments.

Introdução: As infecções respiratórias causam elevadas morbidade e mortalidade, sendo os vírus os principais agentes destas doenças. O monitoramento e vigilância de vírus respiratórios, desde os mais conhecidos até os emergentes, são importantes para a gestão em saúde, orientando tempo de profilaxia e minimizando o impacto de epidemias nas comunidades. Objetivos: Estudar a epidemiologia molecular do vírus sincicial respiratório (VSR) e descrever a epidemiologia dos seguintes vírus: influenza (IF), influenza A (H1N1), adenovírus (AdV) e parainfluenza (PIV) no Hospital de Clínicas de Porto Alegre. Para isso foram conduzidos três estudos: (1) caracterização das infecções respiratórias causadas por VSR, IF, AdV e PIV em crianças; (2) validação de uma técnica de reação em cadeia da polimerase em tempo real (RT-PCR) para detecção de VSR A/B e IF A/B e (3) caracterização de genótipos do VRS em crianças com infecções comunitárias e hospitalares. Métodos: No primeiro estudo foram levantadas as seguintes variáveis: casos de infecções respiratórias por VSR, AdV, PIV ou IF e H1N1; internações em enfermarias hospitalares e internações em unidades de terapia intensiva (UTI); infecções hospitalares e taxas de letalidade. As variáveis foram coletadas durante o atendimento de crianças (idade 0-12 anos) no HCPA entre 2007 e 2010. No segundo estudo, os alvos do ensaio de RT-PCR foram: o gene da proteína da matriz de IFA, o gene da hemaglutinina do IFB e o gene da nucleoproteína de RSVA/B. A especificidade do ensaio e seu limite de detecção foram determinados. Uma comparação entre RT-PCR e imunofluorescência indireta foi realizada. No terceiro estudo, 63 isolados de VSR (21 de origem nosocomial e 42 adquiridos na comunidade) foram sequenciados para estabelecer uma análise filogenética deste vírus. Resultados: Cada um dos vírus estudados apresentou um comportamento diferente. O VSR demonstrou ser o principal agente envolvido em infecções nosocomiais. Já os pacientes com AdV, bem como o VSR, apresentaram altas taxas de admissão em UTI em 2007 e 2010. O AdV e o H1N1 tiveram as maiores taxas de letalidade. O ensaio de RT-PCR mostrou-se específico e foi mais sensível do que a imunofluorescência, sendo capaz de detectar co-infecções. Os seguintes limites de detecção foram obtidos: 1 cópia/mL para a IFA, 10 cópias/mL para IFB, 5 cópias/mL para RSVA e 250 cópias/mL para RSVB. A investigação dos genótipos de VSR revelou que todos os isolados VSRA circulantes eram do mesmo grupo filogenético, o genótipo NA1 de origem japonesa. Por outro lado, os isolados VSRB foram distribuídos em grupos diferentes: BA4, POA1, POA2, POA3 e POA4. Este estudo foi o primeiro que descreveu a circulação do genótipo NA1 no Brasil, bem como quatro novos genótipos (POA1, POA2, POA3 e POA4). Conclusão: Os resultados obtidos no primeiro estudo demonstram o impacto das epidemias sazonais de vírus respiratórios. O segundo estudo corroborou relatos da literatura que técnicas moleculares, como RT-PCR, são adequadas para a detecção de vírus respiratórios. O terceiro estudo relatou genótipos emergentes de VSR. Estudos de vigilância como os descritos acima deveriam ser conduzidos periodicamente para acompanhar o padrão de comportamento dos vírus na população alvo. / Background: Respiratory tract infections of viral origin are a major cause of morbidity and mortality worldwide. Surveillance of well-known viruses and emerging threats is important for management, prophylaxis and to minimize impact on the community. Objective: To study the molecular epidemiology of respiratory syncytial virus (RSV) and describe the epidemiology of viruses: influenza (IF), influenza A (H1N1), adenovirus (AdV) and parainfluenza (PIV) at Hospital de Clinicas de Porto Alegre. Three studies were performed: (1) characterization of respiratory infections caused by RSV, IF, AdV and PIV in children, (2) validation of a technique of polymerase chain reaction in real-time (RT-PCR) to detect RSVA/B and IFA/B and (3) detection of genotypes of RSV in children with communityand hospital-acquired infection. Methods: In the first study, variables such as number of cases of viral (RSV, AdV, PIV or IF and H1N1) infection, hospitalizations in general wards and intensive care units (ICUs), nosocomial infections and lethality rates were collected. These variables obtained from each children (age 0-12 years) between 2007 and 2010. In the second study the assay RT-PCR was used to target the matrix gene of IFA, the hemagglutinin gene of IFB and the nucleoprotein gene of RSVA/B. The specificity of the assay and its limit of detection were determined. A comparison of RT-PCR and indirect immunofluorescence was performed. In the third study, 63 RSV isolates (21 nosocomial and 42 community-acquired) were submitted to sequencing to establish a phylogenetic analysis of this virus. Results: The different viruses presented a diversity of behaviors according to hospitalization, nosocomial outbreaks or lethality in children. RSV accounted for most nosocomial infections. Rates of ICU admission for AdV and RSV infection were highest in 2007 and 2010. During 2008–2009, H1N1 and AdV had the highest ICU admission rates. AdV and H1N1 had the highest lethality rates. The RT-PCR assay was more sensitive than immunofluorescence and it was able to detect viral co-infections. Futhermore, the limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The genotyping study showed that hospital- and community-acquired RSVA isolates were from the same phylogenetic group (the same group of the NA1 Japanese isolates). Conversely, RSVB isolates were distributed across different groups: BA4, POA1, POA2, POA3 and POA4. This was the first study to describe circulation of the NA1 genotype in Brazil, as well as four RSVB genotypes: POA1, POA2, POA3 and POA4. Conclusion: The results obtained in the first study demonstrate the impact of seasonal epidemics of respiratory viruses. The second study confirmed that molecular techniques such as RT-PCR, are suitable for the detection of respiratory viruses. The third study reported emerging genotypes of RSV. Surveillance studies such as this should be performed periodically to monitor the behavior pattern of the virus in the target population.

Introdução: As infecções respiratórias causam elevadas morbidade e mortalidade, sendo os vírus os principais agentes destas doenças. O monitoramento e vigilância de vírus respiratórios, desde os mais conhecidos até os emergentes, são importantes para a gestão em saúde, orientando tempo de profilaxia e minimizando o impacto de epidemias nas comunidades. Objetivos: Estudar a epidemiologia molecular do vírus sincicial respiratório (VSR) e descrever a epidemiologia dos seguintes vírus: influenza (IF), influenza A (H1N1), adenovírus (AdV) e parainfluenza (PIV) no Hospital de Clínicas de Porto Alegre. Para isso foram conduzidos três estudos: (1) caracterização das infecções respiratórias causadas por VSR, IF, AdV e PIV em crianças; (2) validação de uma técnica de reação em cadeia da polimerase em tempo real (RT-PCR) para detecção de VSR A/B e IF A/B e (3) caracterização de genótipos do VRS em crianças com infecções comunitárias e hospitalares. Métodos: No primeiro estudo foram levantadas as seguintes variáveis: casos de infecções respiratórias por VSR, AdV, PIV ou IF e H1N1; internações em enfermarias hospitalares e internações em unidades de terapia intensiva (UTI); infecções hospitalares e taxas de letalidade. As variáveis foram coletadas durante o atendimento de crianças (idade 0-12 anos) no HCPA entre 2007 e 2010. No segundo estudo, os alvos do ensaio de RT-PCR foram: o gene da proteína da matriz de IFA, o gene da hemaglutinina do IFB e o gene da nucleoproteína de RSVA/B. A especificidade do ensaio e seu limite de detecção foram determinados. Uma comparação entre RT-PCR e imunofluorescência indireta foi realizada. No terceiro estudo, 63 isolados de VSR (21 de origem nosocomial e 42 adquiridos na comunidade) foram sequenciados para estabelecer uma análise filogenética deste vírus. Resultados: Cada um dos vírus estudados apresentou um comportamento diferente. O VSR demonstrou ser o principal agente envolvido em infecções nosocomiais. Já os pacientes com AdV, bem como o VSR, apresentaram altas taxas de admissão em UTI em 2007 e 2010. O AdV e o H1N1 tiveram as maiores taxas de letalidade. O ensaio de RT-PCR mostrou-se específico e foi mais sensível do que a imunofluorescência, sendo capaz de detectar co-infecções. Os seguintes limites de detecção foram obtidos: 1 cópia/mL para a IFA, 10 cópias/mL para IFB, 5 cópias/mL para RSVA e 250 cópias/mL para RSVB. A investigação dos genótipos de VSR revelou que todos os isolados VSRA circulantes eram do mesmo grupo filogenético, o genótipo NA1 de origem japonesa. Por outro lado, os isolados VSRB foram distribuídos em grupos diferentes: BA4, POA1, POA2, POA3 e POA4. Este estudo foi o primeiro que descreveu a circulação do genótipo NA1 no Brasil, bem como quatro novos genótipos (POA1, POA2, POA3 e POA4). Conclusão: Os resultados obtidos no primeiro estudo demonstram o impacto das epidemias sazonais de vírus respiratórios. O segundo estudo corroborou relatos da literatura que técnicas moleculares, como RT-PCR, são adequadas para a detecção de vírus respiratórios. O terceiro estudo relatou genótipos emergentes de VSR. Estudos de vigilância como os descritos acima deveriam ser conduzidos periodicamente para acompanhar o padrão de comportamento dos vírus na população alvo. / Background: Respiratory tract infections of viral origin are a major cause of morbidity and mortality worldwide. Surveillance of well-known viruses and emerging threats is important for management, prophylaxis and to minimize impact on the community. Objective: To study the molecular epidemiology of respiratory syncytial virus (RSV) and describe the epidemiology of viruses: influenza (IF), influenza A (H1N1), adenovirus (AdV) and parainfluenza (PIV) at Hospital de Clinicas de Porto Alegre. Three studies were performed: (1) characterization of respiratory infections caused by RSV, IF, AdV and PIV in children, (2) validation of a technique of polymerase chain reaction in real-time (RT-PCR) to detect RSVA/B and IFA/B and (3) detection of genotypes of RSV in children with communityand hospital-acquired infection. Methods: In the first study, variables such as number of cases of viral (RSV, AdV, PIV or IF and H1N1) infection, hospitalizations in general wards and intensive care units (ICUs), nosocomial infections and lethality rates were collected. These variables obtained from each children (age 0-12 years) between 2007 and 2010. In the second study the assay RT-PCR was used to target the matrix gene of IFA, the hemagglutinin gene of IFB and the nucleoprotein gene of RSVA/B. The specificity of the assay and its limit of detection were determined. A comparison of RT-PCR and indirect immunofluorescence was performed. In the third study, 63 RSV isolates (21 nosocomial and 42 community-acquired) were submitted to sequencing to establish a phylogenetic analysis of this virus. Results: The different viruses presented a diversity of behaviors according to hospitalization, nosocomial outbreaks or lethality in children. RSV accounted for most nosocomial infections. Rates of ICU admission for AdV and RSV infection were highest in 2007 and 2010. During 2008–2009, H1N1 and AdV had the highest ICU admission rates. AdV and H1N1 had the highest lethality rates. The RT-PCR assay was more sensitive than immunofluorescence and it was able to detect viral co-infections. Futhermore, the limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The genotyping study showed that hospital- and community-acquired RSVA isolates were from the same phylogenetic group (the same group of the NA1 Japanese isolates). Conversely, RSVB isolates were distributed across different groups: BA4, POA1, POA2, POA3 and POA4. This was the first study to describe circulation of the NA1 genotype in Brazil, as well as four RSVB genotypes: POA1, POA2, POA3 and POA4. Conclusion: The results obtained in the first study demonstrate the impact of seasonal epidemics of respiratory viruses. The second study confirmed that molecular techniques such as RT-PCR, are suitable for the detection of respiratory viruses. The third study reported emerging genotypes of RSV. Surveillance studies such as this should be performed periodically to monitor the behavior pattern of the virus in the target population.

Introdução: As infecções respiratórias causam elevadas morbidade e mortalidade, sendo os vírus os principais agentes destas doenças. O monitoramento e vigilância de vírus respiratórios, desde os mais conhecidos até os emergentes, são importantes para a gestão em saúde, orientando tempo de profilaxia e minimizando o impacto de epidemias nas comunidades. Objetivos: Estudar a epidemiologia molecular do vírus sincicial respiratório (VSR) e descrever a epidemiologia dos seguintes vírus: influenza (IF), influenza A (H1N1), adenovírus (AdV) e parainfluenza (PIV) no Hospital de Clínicas de Porto Alegre. Para isso foram conduzidos três estudos: (1) caracterização das infecções respiratórias causadas por VSR, IF, AdV e PIV em crianças; (2) validação de uma técnica de reação em cadeia da polimerase em tempo real (RT-PCR) para detecção de VSR A/B e IF A/B e (3) caracterização de genótipos do VRS em crianças com infecções comunitárias e hospitalares. Métodos: No primeiro estudo foram levantadas as seguintes variáveis: casos de infecções respiratórias por VSR, AdV, PIV ou IF e H1N1; internações em enfermarias hospitalares e internações em unidades de terapia intensiva (UTI); infecções hospitalares e taxas de letalidade. As variáveis foram coletadas durante o atendimento de crianças (idade 0-12 anos) no HCPA entre 2007 e 2010. No segundo estudo, os alvos do ensaio de RT-PCR foram: o gene da proteína da matriz de IFA, o gene da hemaglutinina do IFB e o gene da nucleoproteína de RSVA/B. A especificidade do ensaio e seu limite de detecção foram determinados. Uma comparação entre RT-PCR e imunofluorescência indireta foi realizada. No terceiro estudo, 63 isolados de VSR (21 de origem nosocomial e 42 adquiridos na comunidade) foram sequenciados para estabelecer uma análise filogenética deste vírus. Resultados: Cada um dos vírus estudados apresentou um comportamento diferente. O VSR demonstrou ser o principal agente envolvido em infecções nosocomiais. Já os pacientes com AdV, bem como o VSR, apresentaram altas taxas de admissão em UTI em 2007 e 2010. O AdV e o H1N1 tiveram as maiores taxas de letalidade. O ensaio de RT-PCR mostrou-se específico e foi mais sensível do que a imunofluorescência, sendo capaz de detectar co-infecções. Os seguintes limites de detecção foram obtidos: 1 cópia/mL para a IFA, 10 cópias/mL para IFB, 5 cópias/mL para RSVA e 250 cópias/mL para RSVB. A investigação dos genótipos de VSR revelou que todos os isolados VSRA circulantes eram do mesmo grupo filogenético, o genótipo NA1 de origem japonesa. Por outro lado, os isolados VSRB foram distribuídos em grupos diferentes: BA4, POA1, POA2, POA3 e POA4. Este estudo foi o primeiro que descreveu a circulação do genótipo NA1 no Brasil, bem como quatro novos genótipos (POA1, POA2, POA3 e POA4). Conclusão: Os resultados obtidos no primeiro estudo demonstram o impacto das epidemias sazonais de vírus respiratórios. O segundo estudo corroborou relatos da literatura que técnicas moleculares, como RT-PCR, são adequadas para a detecção de vírus respiratórios. O terceiro estudo relatou genótipos emergentes de VSR. Estudos de vigilância como os descritos acima deveriam ser conduzidos periodicamente para acompanhar o padrão de comportamento dos vírus na população alvo. / Background: Respiratory tract infections of viral origin are a major cause of morbidity and mortality worldwide. Surveillance of well-known viruses and emerging threats is important for management, prophylaxis and to minimize impact on the community. Objective: To study the molecular epidemiology of respiratory syncytial virus (RSV) and describe the epidemiology of viruses: influenza (IF), influenza A (H1N1), adenovirus (AdV) and parainfluenza (PIV) at Hospital de Clinicas de Porto Alegre. Three studies were performed: (1) characterization of respiratory infections caused by RSV, IF, AdV and PIV in children, (2) validation of a technique of polymerase chain reaction in real-time (RT-PCR) to detect RSVA/B and IFA/B and (3) detection of genotypes of RSV in children with communityand hospital-acquired infection. Methods: In the first study, variables such as number of cases of viral (RSV, AdV, PIV or IF and H1N1) infection, hospitalizations in general wards and intensive care units (ICUs), nosocomial infections and lethality rates were collected. These variables obtained from each children (age 0-12 years) between 2007 and 2010. In the second study the assay RT-PCR was used to target the matrix gene of IFA, the hemagglutinin gene of IFB and the nucleoprotein gene of RSVA/B. The specificity of the assay and its limit of detection were determined. A comparison of RT-PCR and indirect immunofluorescence was performed. In the third study, 63 RSV isolates (21 nosocomial and 42 community-acquired) were submitted to sequencing to establish a phylogenetic analysis of this virus. Results: The different viruses presented a diversity of behaviors according to hospitalization, nosocomial outbreaks or lethality in children. RSV accounted for most nosocomial infections. Rates of ICU admission for AdV and RSV infection were highest in 2007 and 2010. During 2008–2009, H1N1 and AdV had the highest ICU admission rates. AdV and H1N1 had the highest lethality rates. The RT-PCR assay was more sensitive than immunofluorescence and it was able to detect viral co-infections. Futhermore, the limits of detection were 1 copy/μL for IFA, 10 copies/μL for IFB, 5 copies/μL for RSVA, and 250 copies/μL for RSVB. The genotyping study showed that hospital- and community-acquired RSVA isolates were from the same phylogenetic group (the same group of the NA1 Japanese isolates). Conversely, RSVB isolates were distributed across different groups: BA4, POA1, POA2, POA3 and POA4. This was the first study to describe circulation of the NA1 genotype in Brazil, as well as four RSVB genotypes: POA1, POA2, POA3 and POA4. Conclusion: The results obtained in the first study demonstrate the impact of seasonal epidemics of respiratory viruses. The second study confirmed that molecular techniques such as RT-PCR, are suitable for the detection of respiratory viruses. The third study reported emerging genotypes of RSV. Surveillance studies such as this should be performed periodically to monitor the behavior pattern of the virus in the target population.

Introduction: Epidemiological studies have indicated that 5-38% of influenza like illnesses (ILI) develop into severe disease due to, among others, factors such as; underlying chronic diseases, age, pregnancy, and viral mutations. There are suggestions that dual or multiple virus infections may affect disease severity. This study investigated the association between co-infection between influenza A viruses and other respiratory viruses and disease severity. Methodology: Datum for samples from North West England tested between January 2007 and June 2012 was analysed for patterns of co-infection between influenza A viruses and ten respiratory viruses. Risk of hospitalization to a general ward ICU or death in single versus mixed infections was assessed using multiple logistic regression models. Results: One or more viruses were identified in 37.8% (11,715/30,975) of samples, of which 10.4% (1,214) were mixed infections and 89.6% (10,501) were single infections. Among patients with influenza A(H1N1)pdm09, co-infections occurred in 4.7% (137⁄2,879) vs. 6.5% (59⁄902) in those with seasonal influenza A virus infection. In general, patients with mixed respiratory virus infections had a higher risk of admission to a general ward (OR: 1.43, 95% CI: 1.2 – 1.7, p = <0.0001) than those with a single infection. Co-infection between seasonal influenza A viruses and influenza B virus was associated with a significant increase in the risk of admission to ICU/ death (OR: 22.0, 95% CI: 2.21 – 219.8 p = 0.008). RSV/seasonal influenza A viruses co-infection also associated with increased risk but this was not statistically significant. For the pandemic influenza A(H1N1)pdm09 virus, RSV and AdV co-infection increased risk of hospitalization to a general ward, whereas Flu B increased risk of admission to ICU/ death, but none of these were statistically significant. Considering only single infections, RSV and hPIV1-3 increased risk of admission to a general ward (OR: 1.49, 95% CI: 1.28 – 1.73, p = <0.0001 and OR: 1.34, 95% CI: 1.003 – 1.8, p = 0.05) and admission to ICU/ death (OR: 1.5, 95% CI: 1.20 – 2.0, p = <0.0001 and OR: 1.60, 95% CI: 1.02 – 2.40, p = 0.04). Conclusion: Co-infection is a significant predictor of disease outcome; there is insufficient public health data on this subject as not all samples sent for investigation of respiratory virus infection are tested for all respiratory viruses. Integration of testing for respiratory viruses’ co-infections into routine clinical practice and R&D on integrated drugs and vaccines for influenza A&B, RSV, and AdV, and development of multi-target diagnostic tests is encouraged.