Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The feline calicivirus (FCV) and the felid herpesvirus type 1 (FeHV-1) are the main agents of the respiratory tract diseases of felines. Both viruses are distributed worldwide, however its distribution and prevalence in Brazil are not well known. In the present thesis we describe the isolation and one serosurvey of FCV and FeHV-1 in some counties of the Rio Grande do Sul State, Brazil; and the molecular characterization of the capsid protein gene of FCV isolates is also described. In Chapter 1, we describe the epidemiologic survey of FCV and FeHV-1 through investigation of the conjunctival, nasal, oral and oropharyngeal swabs from 302 domestic cats. The viral isolation was performed in Crandell-Reese feline kidney cells and the isolates were submitted to PCR and RT-PCR for confirmation of the presence of the FeHV-1 and FCV, respectively. Fifty five (18.2%) of the 302 cats analyzed were positive for the viral isolation; when tested by PCR, 29 cats (52.7%) were shedding the FCV, 21 (38.2%) shedding FeHV-1, and 5 cats (9.1%) shedding both viruses. In addition, isolates of FCV and FeHV-1 were standardized to be used as control in the PCR and virus neutralization (VN) assays (serological technical used in chapter 3 of the thesis). The SV65/90 (FCV) and the SV534/00 (FeHV-1) isolates were defined as standard viruses for the assays. In Chapter 2, the molecular characterization of 13 FCV isolates is described; ten isolates came from the survey performed in chapter 1, two were deposited in the virology laboratory of the UFSM and one was obtained from a cat with gingivitis-stomatitis syndrome. The ORF2 (open reading frame) region that codifies the major protein of the viral capsid was sequenced, which includes an immunodominant region of the virus. The ORF 2 was analyzed at the nucleotide and amino acid levels and these data was used for phylogenetic studies of the 13 Brazilian isolates of FCV. These isolates were compared to ten reference strains of FCV and to the San Miguel sea lion virus type 1 (SMSV-1) as an outgroup in phylogenetic study. The primers were designed using the complete sequence of FCV-F9. The comparison of the 13 Brazilian isolates with the F9 strain revealed a large molecular diversity among them, concentrated mainly along the linear epitopes of the region. The Chapter 3 describes a serosurvey against FCV and FeHV-1 in serum samples from domestic feline. From the 630 samples tested by the VN assay, 53.6% (338/630) were positive to one or both viruses with a prevalence of 39.2% for neutralizing antibodies against FCV and 30.6% for FeHV-1. The results from the present study demonstrated that the FCV and FeHV-1 are circulating among the feline population examined, and also, the presence of carriers of the viruses among these cats. Besides, the molecular characterization of the FCV evidenced the great genetic variability of the Brazilian isolates when compared to vaccine and reference strains. / O calicivírus felino (feline calicivirus FCV) e o herpesvírus felino tipo 1 (felid herpesvirus type 1 FeHV-1) são os principais agentes das doenças do trato respiratório dos felinos. Ambos os vírus são mundialmente distribuídos, mas no Brasil sua distribuição e prevalência são ainda pouco conhecidas. Na presente tese descrevemos o isolamento e sorologia do FCV e do FeHV-1 em alguns municípios do estado do Rio Grande do Sul (RS), Brasil; e a caracterização molecular do gene da proteína do capsídeo de isolados de FCV. No Capítulo 1, descreve-se o isolamento do FCV e do FeHV-1 por meio da coleta de suabes (conjuntival, nasal, oral e orofaringeano) de 302 gatos domésticos. O material coletado foi inoculado em cultivo celular de origem felina (CRFK) e submetidos a PCR e RT-PCR para confirmação em FeHV-1 e FCV, respectivamente. Dos 302 gatos analisados, 55 (18,2%) foram positivos no isolamento; e quando testados por PCR, 29 dos 55 gatos (52,7%) excretavam o FCV, 21 (38,2%) excretavam o FeHV-1 e 5 gatos (9,1%) apresentavam ambos os vírus. Além dessa descrição, realizou-se a padronização de um isolado de FCV e de FeHV-1, para serem utilizados como controle nas técnicas de PCR e na vírus neutralização [VN] (técnica sorológica utilizada no capítulo 3 da tese). O SV65/90 (FCV) e o SV534/00 (FeHV-1) foram os isolados definidos como vírus-padrão. No Capítulo 2, incluímos o estudo sobre a análise molecular de 13 isolados de FCV; dez isolados foram obtidos a partir do material descrito no capítulo 1, dois estavam armazenados no laboratório de virologia da UFSM e um foi obtido de um gato acometido pela síndrome gengivo-estomatite. A região analisada foi o gene da proteína do capsídeo, codificado pela ORF2 (open reading frame), na qual se localizam as regiões imunodominantes do FCV. A ORF2 foi analisada em nível de nucleotídeos e aminoácidos; e uma análise filogenética dos 13 isolados brasileiros de FCV, incluindo dez cepas de referência e o vírus dos leões marinhos de San Miguel tipo 1 (San Miguel sea lion virus type 1 SMSV-1) como um outgroup, foi também realizada. Os primers foram desenhados com base na sequência completa do genoma da cepa de referência a FCV-F9. Quando os 13 isolados brasileiros de FCV foram comparados a cepa F9, detectou-se grande diversidade molecular nas regiões variáveis do gene e nos epitopos lineares mapeados. O Capítulo 3 contem um estudo sorológico contra FCV e FeHV-1 em amostras de soro de felinos domésticos. Das 630 amostras testadas pela VN, 53,6% (338/630) foram positivas para um ou ambos os vírus; sendo a prevalência de anticorpos neutralizantes contra o FCV de 39,2% e do FeHV-1 de 30,6%. Os resultados aqui apresentados demonstram a circulação do FCV e do FeHV-1 na população de gatos domésticos analisados, assim como, a presença de gatos portadores para ambos os vírus. Além disso, a caracterização molecular do FCV demonstrou a grande variabilidade genética dos isolados brasileiros em comparação às cepas vacinais e às de referência.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.