RESUMO
Human noroviruses (HuNoVs) are the cause of more than 95% of epidemic non-bacterial gastroenteritis worldwide, with some lethal cases. These viral agents affect people of all ages. However, young children and older adults are the highest-risk groups, being affected with the greatest rate of hospitalizations and morbidity cases. HuNoV structural proteins, especially VP1, have been studied extensively. In contrast, the functions of the non-structural proteins of the virus have been undescribed in depth. Studies on HuNoV non-structural proteins have mostly been made by expressing them individually in in vitro cultures, providing insights of their functions and the role that they play in HuNoV replication and pathogenesis. This review examines exhaustively the functions of both HuNoV structural and non-structural proteins and their possible role within the viral replicative cycle and the pathogenesis of the virus. It also highlights recent findings regarding the host's innate and adaptive immune responses against HuNoV, which are of great relevance for diagnostics and vaccine development so as to prevent infections caused by these fastidious viruses.
Assuntos
Imunidade Adaptativa , Infecções por Caliciviridae/virologia , Imunidade Inata , Norovirus/patogenicidade , Proteínas Virais/metabolismo , Replicação Viral , Animais , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Norovirus/crescimento & desenvolvimento , Norovirus/imunologia , Norovirus/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/imunologia , VirulênciaRESUMO
Noroviruses (NoV) cause the majority of non-bacterial gastroenteritis cases worldwide, with genotype II.4 being the most common. The aim of our study was to quantitate norovirus-specific IgG in immunocompromised patients before and after laboratory-confirmed norovirus infection. A quantitative ELISA was developed by coating ELISA plates with recombinantly expressed P domain of GII.1 capsid protein. After testing mouse sera drawn before and after immunization with GII.1- and GII.4 P domain, sera from GII.1- and GII.4 infected patients were tested. The assay reliably detected preexisting NoV-specific IgG antibodies. Sera drawn after infection showed increased antibody concentrations. Antibodies elicited by GII.1- and GII.4 infections could be detected with coated GII.1 capsid protein. IgG levels remained constant during the first week and then increased in the second week after laboratory diagnosis. The results show that immunocompromised patients elicited IgG responses to NoV infections that could be reliably detected with our quantitative ELISA.
Assuntos
Anticorpos Antivirais/sangue , Infecções por Caliciviridae/imunologia , Hospedeiro Imunocomprometido , Imunoglobulina G/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , NorovirusRESUMO
Noroviruses and Sapoviruses, classified in the Caliciviridae family, are small positive-stranded RNA viruses, considered nowadays the leading cause of acute gastroenteritis globally in both children and adults. Although most noroviruses have been associated with gastrointestinal disease in humans, almost 50 years after its discovery, there is still a lack of comprehensive evidence regarding its biology and pathogenesis mainly because they can be neither conveniently grown in cultured cells nor propagated in animal models. However, other members of this family such as Feline calicivirus (FCV), Murine norovirus (MNV), Rabbit hemorrhagic disease virus (RHDV), and Porcine sapovirus (PS), from which there are accessible propagation systems, have been useful to study the calicivirus replication strategies. Using cell cultures and animal models, many of the functions of the viral proteins in the viral replication cycles have been well-characterized. Moreover, evidence of the role of viral proteins from different members of the family in the establishment of infection has been generated and the mechanism of their immunopathogenesis begins to be understood. In this review, we discuss different aspects of how caliciviruses are implicated in membrane rearrangements, apoptosis, and evasion of the immune responses, highlighting some of the pathogenic mechanisms triggered by different members of the Caliciviridae family.
Assuntos
Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Caliciviridae/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade , Imunomodulação , Imunidade Adaptativa , Animais , Peptídeos Catiônicos Antimicrobianos , Apoptose , Caliciviridae/genética , Infecções por Caliciviridae/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Efeito Citopatogênico Viral , Suscetibilidade a Doenças , Regulação Viral da Expressão Gênica , Genoma Viral , Humanos , Evasão da Resposta Imune , Imunidade Inata , Interações Microbianas , Microbiota , Replicação ViralRESUMO
Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis, including both outbreaks and endemic infections. The development of preventive strategies, including vaccines, for the most susceptible groups (children <5years of age, the elderly and individuals suffering crowding, such as military personnel and travelers) is desirable. However, NoV vaccine development has faced many difficulties, including genetic/antigenic diversity, limited knowledge on NoV immunology and viral cycle, lack of a permissive cell line for cultivation and lack of a widely available and successful animal model. Vaccine candidates rely on inoculation of virus-like particles (VLPs) formed by the main capsid protein VP1, subviral particles made from the protruding domain of VP1 (P-particles) or viral vectors with a NoV capsid gene insert produced by bioengineering technologies. Polivalent vaccines including multiple NoV genotypes and/or other viruses acquired by the enteric route have been developed. A VLP vaccine candidate has reached phase II clinical trials and several others are in pre-clinical stages of development. In this article we discuss the main challenges facing the development of a NoV vaccine and the current status of prevailing candidates.
Assuntos
Norovirus/patogenicidade , Vacinas Virais/uso terapêutico , Doença Aguda , Animais , Bioengenharia/métodos , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/prevenção & controle , Proteínas do Capsídeo/imunologia , Gastroenterite/imunologia , Gastroenterite/prevenção & controle , Humanos , Norovirus/imunologiaRESUMO
Diarrheal diseases (DD) have distinct etiological profiles in immune-deficient and immune-competent patients. This study compares detection rates, genotype distribution and viral loads of different enteric viral agents in HIV-1 seropositive (n = 200) and HIV-1 seronegative (n = 125) children hospitalized with DD in Rio de Janeiro, Brazil. Except for group A rotavirus (RVA), which were detected through enzyme immunoassay, the other enteric viruses (norovirus [NoV], astrovirus [HAstV], adenovirus [HAdV] and bocavirus [HBoV]) were detected through PCR or RT-PCR. A quantitative PCR was performed for RVA, NoV, HAstV, HAdV and HBoV. Infections with NoV (19% vs. 9.6%; p<0.001), HBoV (14% vs. 7.2%; p = 0.042) and HAdV (30.5% vs. 14.4%; p<0.001) were significantly more frequent among HIV-1 seropositive children. RVA was significantly less frequent among HIV-1 seropositive patients (6.5% vs. 20%; p<0.001). Similarly, frequency of infection with HAstV was lower among HIV-1 seropositive children (5.5% vs. 12.8%; p = 0.018). Among HIV-1 seropositive children 33 (16.5%) had co-infections, including three enteric viruses, such as NoV, HBoV and HAdV (n = 2) and NoV, HAstV and HAdV (n = 2). The frequency of infection with more than one virus was 17 (13.6%) in the HIV-1 negative group, triple infection (NoV + HAstV + HBoV) being observed in only one patient. The median viral load of HAstV in feces was significantly higher among HIV-1 positive children compared to HIV-1 negative children. Concerning children infected with RVA, NoV, HBoV and HAdV, no statistically significant differences were observed in the medians of viral loads in feces, comparing HIV-1 seropositive and HIV-1 seronegative children. Similar detection rates were observed for RVA, HAstV and HAdV, whilst NoV and HBoV were significantly more prevalent among children with CD4+ T lymphocyte count below 200 cells/mm3. Enteric viruses should be considered an important cause of DD in HIV-1 seropositive children, along with pathogens more classically associated with intestinal infections in immunocompromised hosts.
Assuntos
Infecções por Adenoviridae/epidemiologia , Infecções por Astroviridae/epidemiologia , Infecções por Caliciviridae/epidemiologia , Diarreia/epidemiologia , Gastroenterite/epidemiologia , Infecções por HIV/epidemiologia , Infecções por Parvoviridae/epidemiologia , Infecções por Rotavirus/epidemiologia , Adenoviridae/crescimento & desenvolvimento , Adenoviridae/isolamento & purificação , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/virologia , Infecções por Astroviridae/imunologia , Infecções por Astroviridae/virologia , Brasil/epidemiologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Criança , Pré-Escolar , Coinfecção , Diarreia/imunologia , Diarreia/virologia , Fezes/virologia , Feminino , Gastroenterite/imunologia , Gastroenterite/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/isolamento & purificação , Bocavirus Humano/crescimento & desenvolvimento , Bocavirus Humano/isolamento & purificação , Humanos , Lactente , Masculino , Mamastrovirus/crescimento & desenvolvimento , Mamastrovirus/isolamento & purificação , Norovirus/crescimento & desenvolvimento , Norovirus/isolamento & purificação , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , Prevalência , Rotavirus/crescimento & desenvolvimento , Rotavirus/isolamento & purificação , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/virologia , Carga ViralRESUMO
Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans.
Assuntos
Infecções por Caliciviridae/prevenção & controle , Vírus da Doença Hemorrágica de Coelhos/imunologia , Conformação Molecular , Pichia/metabolismo , Temperatura , Vacinas Virais/biossíntese , Vírion/imunologia , Sequência de Aminoácidos , Animais , Soluções Tampão , Infecções por Caliciviridae/imunologia , Cromatografia em Gel , Peste Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Vírus da Febre Suína Clássica/imunologia , Resposta ao Choque Térmico , Hemaglutinação , Concentração de Íons de Hidrogênio , Imunização , Dados de Sequência Molecular , Concentração Osmolar , Peptídeos/química , Peptídeos/imunologia , Coelhos , Sefarose , Suínos , Vírion/ultraestrutura , ViscosidadeRESUMO
Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.
Assuntos
Infecções por Caliciviridae/veterinária , Vetores Genéticos/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Coelhos/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Adenoviridae/genética , Administração através da Mucosa , Animais , Anticorpos Antivirais/sangue , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/prevenção & controle , Infecções por Caliciviridae/virologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Vetores Genéticos/genética , Testes de Inibição da Hemaglutinação/veterinária , Vírus da Doença Hemorrágica de Coelhos/genética , Imunização/métodos , Imunização/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Coelhos/virologia , Distribuição Aleatória , Estatísticas não Paramétricas , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Vitamin A supplementation is associated with divergent clinical norovirus (NoV) outcomes in Mexican children. Fecal cytokine concentrations following NoV genogroup infections among 127 Mexican children 5-15 mo old enrolled in a randomized, double-blind, placebo-controlled, vitamin A supplementation trial were determined to clarify the role the gut immune response plays in these associations. Stools collected from supplemented children [20,000 IU retinol (3.3 IU = 1 µg retinol) for children < 12 mo of age; 45,000 iu for children ≥ 12 mo] or children in the placebo group were screened for NoV genogroups I (GI) and II (GII). Monocyte chemoattractant protein-1 (MCP-1), TNFα, IL-5, IL-6, IL-8, IL-4, IFNγ, and IL-10 fecal concentrations were also determined. Differences in cytokine levels between the 2 groups following GI and GII infections were determined using ordered logistic regression models. MCP-1 and IL-8 levels were greater among GI- and GII-infected children, respectively, compared with uninfected children, whereas IL-5 levels were greater following both genogroup infections. MCP-1, IL-8, and IL-6 fecal levels were reduced among supplemented children with GII-associated diarrhea compared with the placebo group. Vitamin A-supplemented, GII-infected children had reduced MCP-1 and TNFα levels compared with GII-infected children in the placebo group (P-interaction = 0.02 and 0.03, respectively). Supplemented children with GI-associated diarrhea had higher TNFα and IL-4 levels compared with children in the placebo group with diarrhea (P-interaction = 0.02 and 0.02, respectively). The divergent effects of supplementation on NoV outcomes may result from the different effects vitamin A has on the genogroup-specific immune responses.
Assuntos
Infecções por Caliciviridae/prevenção & controle , Quimiocinas/análise , Citocinas/análise , Interações Hospedeiro-Patógeno , Intestinos/imunologia , Norovirus/fisiologia , Vitamina A/uso terapêutico , Imunidade Adaptativa , Infecções por Caliciviridae/imunologia , Quimiocinas/imunologia , Citocinas/imunologia , Suplementos Nutricionais , Método Duplo-Cego , Fezes/química , Fezes/microbiologia , Feminino , Gastroenterite/imunologia , Gastroenterite/prevenção & controle , Humanos , Imunidade Inata , Imunomodulação , Lactente , Intestinos/microbiologia , Masculino , México , Norovirus/classificação , Norovirus/imunologia , Norovirus/isolamento & purificação , Deficiência de Vitamina A/imunologia , Deficiência de Vitamina A/prevenção & controleRESUMO
Rabbit hemorrhagic disease virus (RHDV) VP60 capsid protein was recently expressed at approximately 1.5 gL(-1) associated with the disruption pellet of Pichia pastoris, thus requiring an additional process of extraction by solubilization. Consequently, the expression of a soluble variant of VP60 was undertaken in order to attain an easier approach for vaccine production. The VP60 gene was cloned without secretion signal under the transcriptional control of the AOX1 yeast promoter. The antigen obtained was intracellular and soluble at approximately 480 mg L(-1). Its characterization by size-exclusion HPLC, ultracentrifugation, and electron microscopy, showed the presence of high molecular weight structures similar in mass, size and buoyant density to native RHDV. The antigenic profile was similar to that from authentic virions as determined with monoclonal antibodies directed against RHDV conformational epitopes. These analyses, conducted on VP60 obtained insoluble in P. pastoris revealed the formation of protein aggregates rather than the presence of ordered multimeric structures. An immunization trial was conducted in which the soluble VP60 was employed by subcutaneous (s.c.) injection either purified by a single chromatographic step or contained within raw disruption supernatant, emulsified in Montanide 888. The insoluble variant was administered as a yeast extract powder by oral and s.c. routes. The earliest IgG response, titers and persistence of antibodies were studied by competition ELISA and hemagglutination inhibition (HI) assays. All rabbits immunized with the yeast-derived antigens developed a strong RHDV-specific response (including the "RHDVa" subtype) that lasted over one year after the primary immunization. Early HI titers up to 1/40 960 were generated. The immune response was similar to that induced by VP60 from Sf9 cells and superior to the response elicited with inactivated RHDV. Overall it was found that the soluble VP60 multimers, safely and easily produced in P. pastoris, are a valuable candidate for the rational implementation of a low-cost, scalable subunit vaccine against RHDV.
Assuntos
Infecções por Caliciviridae/veterinária , Proteínas do Capsídeo/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Pichia/metabolismo , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/prevenção & controle , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Expressão Gênica , Vírus da Doença Hemorrágica de Coelhos/genética , Imunização/veterinária , Imunoglobulina G/sangue , Pichia/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Vacinas Virais/economia , Vacinas Virais/genética , Vacinas Virais/isolamento & purificaçãoRESUMO
Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004-2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region.