RESUMEN
Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.
Asunto(s)
Infecciones por Alphavirus , Alphavirus , Genotipo , Macaca fascicularis , ARN Viral , Viremia , Animales , Macaca fascicularis/virología , Alphavirus/genética , Alphavirus/patogenicidad , Alphavirus/clasificación , Alphavirus/aislamiento & purificación , Infecciones por Alphavirus/virología , Infecciones por Alphavirus/veterinaria , Viremia/virología , ARN Viral/genética , Anticuerpos Antivirales/sangre , Anticuerpos Neutralizantes/sangre , Modelos Animales de Enfermedad , Filogenia , Citocinas/genética , Citocinas/sangreRESUMEN
Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.
Asunto(s)
Infecciones por Alphavirus , Alphavirus , Genoma Viral , Proteínas no Estructurales Virales , Replicación Viral , Replicación Viral/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Animales , Alphavirus/genética , Alphavirus/patogenicidad , Ratones , Infecciones por Alphavirus/virología , Genoma Viral/genética , Línea Celular , Humanos , AustraliaRESUMEN
The impact of the host microbiota on arbovirus infections is currently not well understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that preincubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivity in vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of the Alphavirus genus and Zika virus of the Flavivirus genus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after preincubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses. IMPORTANCE During the past decades, the world has experienced a vast increase in epidemics of alphavirus and flavivirus infections. These viruses can cause severe diseases, such as hemorrhagic fever, encephalitis, and arthritis. Several alpha- and flaviviruses, such as chikungunya virus, Zika virus, and dengue virus, are significant global health threats because of their high disease burden, their widespread (re-)emergence, and the lack of (good) anti-arboviral strategies. Despite the clear health burden, alphavirus and flavivirus infection and disease are not fully understood. A knowledge gap in the interplay between the host and the arbovirus is the potential interaction with host skin bacteria. Therefore, we studied the effect of (skin) bacteria and bacterial cell wall components on alphavirus and flavivirus infectivity in cell culture. Our results show that certain bacterial cell wall components markedly reduced viral infectivity by interacting directly with the virus particle.
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Alphavirus , Arbovirus , Pared Celular , Flavivirus , Alphavirus/patogenicidad , Alphavirus/fisiología , Animales , Arbovirus/patogenicidad , Arbovirus/fisiología , Bacterias , Virus Chikungunya , Flavivirus/patogenicidad , Flavivirus/fisiología , Lipopolisacáridos , Microbiota , Virus ZikaRESUMEN
The emergence of new epidemic variants of alphaviruses poses a public health risk. It is associated with adaptive mutations that often cause increased pathogenicity. Getah virus (GETV), a neglected and re-emerging mosquito-borne alphavirus, poses threat to many domestic animals and probably even humans. At present, the underlying mechanisms of GETV pathogenesis are not well defined. We identified a residue in the E2 glycoprotein that is critical for viral adsorption to cultured cells and pathogenesis in vivo. Viruses containing an arginine instead of a lysine at residue 253 displayed enhanced infectivity in mammalian cells and diminished virulence in a mouse model of GETV disease. Experiments in cell culture show that heparan sulfate (HS) is a new attachment factor for GETV, and the exchange Lys253Arg improves virus attachment by enhancing binding to HS. The mutation also results in more effective binding to glycosaminoglycan (GAG), linked to low virulence due to rapid virus clearance from the circulation. Localization of residue 253 in the three-dimensional structure of the spike revealed several other basic residues in E2 and E1 in close vicinity that might constitute an HS-binding site different from sites previously identified in other alphaviruses. Overall, our study reveals that HS acts as the attachment factor of GETV and provides convincing evidence for an HS-binding determinant at residue 253 in the E2 glycoprotein of GETV, which contributes to infectivity and virulence. IMPORTANCE Due to decades of inadequate monitoring and lack of vaccines and specific treatment, a large number of people have been infected with alphaviruses. GETV is a re-emerging alphavirus that has the potential to infect humans. This specificity of the GETV disease, particularly its propensity for chronic musculoskeletal manifestations, underscores the need to identify the genetic determinants that govern GETV virulence in the host. Using a mouse model, we show that a single amino acid substitution at residue 253 in the E2 glycoprotein causes attenuation of the virus. Residue 253 might be part of a binding site for HS, a ubiquitous attachment factor on the cell surface. The substitution of Lys by Arg improves cell attachment of the virus in vitro and virus clearance from the blood in vivo by enhancing binding to HS. In summary, we have identified HS as a new attachment factor for GETV and the corresponding binding site in the E2 protein for the first time. Our research potentially improved understanding of the pathogenic mechanism of GETV and provided a potential target for the development of new attenuated vaccines and antiviral drugs.
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Infecciones por Alphavirus , Alphavirus , Sustitución de Aminoácidos , Proteínas del Envoltorio Viral , Alphavirus/genética , Alphavirus/patogenicidad , Infecciones por Alphavirus/virología , Animales , Sitios de Unión/genética , Células Cultivadas , Modelos Animales de Enfermedad , Heparitina Sulfato/metabolismo , Humanos , Ratones , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.
Asunto(s)
Infecciones por Alphavirus/virología , Ganglios Linfáticos/citología , Receptores Inmunológicos/metabolismo , Viremia/patología , Alphavirus/patogenicidad , Animales , Fiebre Chikungunya/genética , Fiebre Chikungunya/virología , Células Endoteliales/virología , Interacciones Huésped-Patógeno , Macrófagos del Hígado/virología , Ganglios Linfáticos/virología , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , ARN Viral/metabolismo , Receptores Inmunológicos/genética , Análisis de la Célula Individual , Viremia/virologíaRESUMEN
Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.
Asunto(s)
Infecciones por Alphavirus/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos ampliamente neutralizantes/inmunología , Alphavirus/inmunología , Alphavirus/patogenicidad , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Artritis/etiología , Artritis/inmunología , Artritis/virología , Anticuerpos ampliamente neutralizantes/aislamiento & purificación , Anticuerpos ampliamente neutralizantes/farmacología , Fiebre Chikungunya/virología , Virus Chikungunya/inmunología , Virus Chikungunya/patogenicidad , Reacciones Cruzadas , Epítopos/inmunología , Células Germinativas/inmunología , Glicoproteínas/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Human pathogens belonging to the Alphavirus genus, in the Togaviridae family, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.
Asunto(s)
Infecciones por Alphavirus , Alphavirus , Alphavirus/inmunología , Alphavirus/patogenicidad , Infecciones por Alphavirus/epidemiología , Infecciones por Alphavirus/prevención & control , Infecciones por Alphavirus/virología , Animales , Humanos , Vacunas Virales/inmunologíaRESUMEN
Virus-induced infections of the central nervous system (CNS) are among the most serious problems in public health and can be associated with high rates of morbidity and mortality, mainly in low- and middle-income countries, where these manifestations have been neglected. Typically, herpes simplex virus 1 and 2, varicella-zoster, and enterovirus are responsible for a high number of cases in immunocompetent hosts, whereas other herpesviruses (for example, cytomegalovirus) are the most common in immunocompromised individuals. Arboviruses have also been associated with outbreaks with a high burden of neurological disorders, such as the Zika virus epidemic in Brazil. There is a current lack of understanding in Brazil about the most common viruses involved in CNS infections. In this review, we briefly summarize the most recent studies and findings associated with the CNS, in addition to epidemiological data that provide extensive information on the circulation and diversity of the most common neuro-invasive viruses in Brazil. We also highlight important aspects of the prion-associated diseases. This review provides readers with better knowledge of virus-associated CNS infections. A deeper understanding of these infections will support the improvement of the current surveillance strategies to allow the timely monitoring of the emergence/re-emergence of neurotropic viruses.
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Enfermedades del Sistema Nervioso Central/virología , Infecciones del Sistema Nervioso Central/epidemiología , Enfermedades por Prión/epidemiología , Alphavirus/patogenicidad , Brasil/epidemiología , Sistema Nervioso Central/virología , Enfermedades del Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central/fisiopatología , Infecciones del Sistema Nervioso Central/virología , Enfermedades Virales del Sistema Nervioso Central/fisiopatología , Enfermedades Virales del Sistema Nervioso Central/virología , Enterovirus/patogenicidad , Flavivirus/patogenicidad , Herpesviridae/patogenicidad , Humanos , Enfermedades del Sistema Nervioso/epidemiología , Enfermedades del Sistema Nervioso/virología , Enfermedades por Prión/fisiopatología , Priones/metabolismo , Priones/patogenicidad , Simplexvirus/patogenicidad , Virosis/virología , Virus/patogenicidad , Virus Zika/patogenicidadRESUMEN
Mayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.
Asunto(s)
Infecciones por Alphavirus/diagnóstico , Alphavirus/inmunología , Epítopos/inmunología , Infecciones por Togaviridae/diagnóstico , Aedes/virología , Alphavirus/patogenicidad , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/transmisión , Infecciones por Alphavirus/virología , Animales , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Epítopos/ultraestructura , Femenino , Genes Sintéticos/genética , Genes Sintéticos/inmunología , Humanos , Inmunoglobulina M/inmunología , Masculino , Pruebas Serológicas , América del Sur/epidemiología , Togaviridae/aislamiento & purificación , Togaviridae/patogenicidad , Infecciones por Togaviridae/inmunología , Infecciones por Togaviridae/transmisión , Infecciones por Togaviridae/virologíaRESUMEN
Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3 Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.
Asunto(s)
Infecciones por Alphavirus/prevención & control , Fiebre Chikungunya/prevención & control , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismo , Alphavirus/patogenicidad , Infecciones por Alphavirus/metabolismo , Infecciones por Alphavirus/virología , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Fiebre Chikungunya/metabolismo , Fiebre Chikungunya/virología , Virus Chikungunya/patogenicidad , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/fisiología , Proteínas de Unión al ARN/fisiología , Internalización del VirusRESUMEN
While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)-a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase-deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.
Asunto(s)
Alphavirus/genética , ADN Helicasas/genética , N-Glicosil Hidrolasas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas no Estructurales Virales/genética , Alphavirus/patogenicidad , Animales , Artritis/virología , Culicidae/virología , Encefalitis/virología , Exantema/virología , Regulación Viral de la Expresión Génica/genética , Células HeLa , Humanos , Proteínas de Unión al ARN/genéticaRESUMEN
Mayaro fever is an infection caused by Mayaro virus (MAYV) that stands out among the neglected diseases transmitted by arthropods. Brazil is the country with the highest number of confirmed cases of MAYV infection. However, epidemiological surveillance studies conducted in Brazil are decentralized and focus on small outbreaks and unconfirmed cases. Thus, the aim of this review was to determine the general epidemiological profile of MAYV infections in Brazil. Several medical databases (i.e., PUBMED/MEDLINE, Scopus, Cochrane Library, LILACS, SciELO, and Biblioteca Virtual em Saúde) were searched for studies reporting cases of MAYV infections in Brazilian patients. Then, the rate of exposure to MAYV in Brazil was analyzed using RStudio® Software. We identified 37 studies published from 1957 to 2019, containing data of 12,374 patients from 1955 to 2018. The general rate of exposure to MAYV in Brazil was 10% (95% CI; 0.04-0.22), with 1,304 reported cases. The highest incidence of MAYV infection was found in the northern region (13%; 95% CI; 0.05-0.29), with 1,142 cases (88% of all cases). Furthermore, autochthonous MAYV cases have also been reported in the Central West (8%; 95% CI; 0.03-0.18) and Southeast (0.4%; 95% CI; 0.00-0.28). The states with the highest number of cases are Amazonas (490 cases), Pará (276 cases), and Goiás (87 cases). In conclusion, the general rate of exposure to MAYV in Brazil between 1955 and 2018 was considerable, especially in the Legal Amazon, in which 93% of cases were reported.
Asunto(s)
Infecciones por Alphavirus/epidemiología , Infecciones por Alphavirus/virología , Alphavirus/patogenicidad , Animales , Brasil/epidemiología , Brotes de Enfermedades , HumanosRESUMEN
Emerging and re-emerging arthritogenic alphaviruses, such as Chikungunya virus (CHIKV) and O'nyong nyong virus, cause acute and chronic crippling arthralgia associated with inflammatory immune responses. Approximately 50% of CHIKV-infected patients suffer from rheumatic manifestations that last 6 months to years. However, the physiological functions of individual immune signaling pathways in the pathogenesis of alphaviral arthritis remain poorly understood. Here, we report that a deficiency in CXCL10, which is a chemoattractant for monocytes/macrophages/T cells, led to the same viremia as wild-type animals, but fewer immune infiltrates and lower viral loads in footpads at the peak of arthritic disease (6-8 days post infection). Macrophages constituted the largest immune cell population in footpads following infection, and were significantly reduced in Cxcl10-/- mice. The viral RNA loads in neutrophils and macrophages were reduced in Cxcl10-/- compared to wild-type mice. In summary, our results demonstrate that CXCL10 signaling promotes the pathogenesis of alphaviral disease and suggest that CXCL10 may be a therapeutic target for mitigating alphaviral arthritis.
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Infecciones por Alphavirus/inmunología , Alphavirus/patogenicidad , Artritis Infecciosa/inmunología , Quimiocina CXCL10/inmunología , Transducción de Señal/inmunología , Alphavirus/genética , Infecciones por Alphavirus/fisiopatología , Animales , Artritis Infecciosa/virología , Quimiocina CXCL10/genética , Modelos Animales de Enfermedad , Femenino , Macrófagos/inmunología , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/virología , Carga Viral , Viremia/inmunologíaRESUMEN
B cell responses are a crucial part of the adaptive immune response to viral infection. Infection by salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) in Atlantic salmon (Salmo salar) and is a serious concern to the aquaculture industry. In this study, we have used intraperitoneal (IP) infection with SAV3 as a model to characterize local B cell responses in the peritoneal cavity (PerC) and systemic immune tissues (head kidney/spleen). Intraperitoneal administration of vaccines is common in Atlantic salmon and understanding more about the local PerC B cell response is fundamental. Intraperitoneal SAV3 infection clearly induced PerC B cell responses as assessed by increased frequency of IgM+ B cells and total IgM secreting cells (ASC). These PerC responses were prolonged up to nine weeks post-infection and positively correlated to the anti-SAV3 E2 and to neutralizing antibody responses in serum. For the systemic immune sites, virus-induced changes in B cell responses were more modest or decreased compared to controls in the same period. Collectively, data reported herein indicated that PerC could serve as a peripheral immunological site by providing a niche for prolonged maintenance of the ASC response in Atlantic salmon.
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Inmunidad Adaptativa , Infecciones por Alphavirus/veterinaria , Alphavirus/patogenicidad , Linfocitos B/virología , Enfermedades de los Peces/virología , Inmunidad Humoral , Salmo salar/virología , Alphavirus/inmunología , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/metabolismo , Infecciones por Alphavirus/virología , Animales , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/metabolismo , Interacciones Huésped-Patógeno , Cavidad Peritoneal , Salmo salar/inmunología , Salmo salar/metabolismoRESUMEN
Salmonid alphavirus (SAV) is the cause of pancreas disease and sleeping disease in farmed salmonid fish in Europe. The spread of these diseases has been difficult to control with biosecurity and current vaccination strategies, and increased understanding of the viral pathogenesis could be beneficial for the development of novel vaccine strategies. N-glycosylation of viral envelope proteins may be crucial for viral virulence and a possible target for its purposed attenuation. In this study, we mutated the N-glycosylation consensus motifs of the E1 and E2 glycoproteins of a SAV3 infectious clone using site-directed mutagenesis. Mutation of the glycosylation motif in E1 gave a complete inactivation of the virus as no viral replication could be detected in cell culture and infectious particles could not be rescued. In contrast, infectious virus particles could be recovered from the SAV3 E2 mutants (E2319Q, E2319A), but not if they were accompanied by lack of N-glycosylation in E1. Compared to the non-mutated infectious clone, the SAV3-E2319Q and SAV3-E2319A recombinant viruses produced less cytopathic effects in cell culture and lower amounts of infectious viral particles. In conclusion, the substitution in the N-linked glycosylation site in E2 attenuated SAV3 in cell culture. The findings could be useful for immunization strategies using live attenuated vaccines and testing in fish will be desirable to study the clone's properties in vivo.
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Alphavirus/genética , Alphavirus/patogenicidad , Salmón/virología , Trucha/virología , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Efecto Citopatogénico Viral/genética , Enfermedades de los Peces/virología , Glicosilación , Mutación/genética , Vacunas Atenuadas , Proteínas del Envoltorio Viral/metabolismo , Virulencia/genéticaRESUMEN
The identification of host / pathogen interactions is essential to both understanding the molecular biology of infection and developing rational intervention strategies to overcome disease. Alphaviruses, such as Sindbis virus, Chikungunya virus, and Venezuelan Equine Encephalitis virus are medically relevant positive-sense RNA viruses. As such, they must interface with the host machinery to complete their infectious lifecycles. Nonetheless, exhaustive RNA:Protein interaction discovery approaches have not been reported for any alphavirus species. Thus, the breadth and evolutionary conservation of host interactions on alphaviral RNA function remains a critical gap in the field. Herein we describe the application of the Cross-Link Assisted mRNP Purification (CLAMP) strategy to identify conserved alphaviral interactions. Through comparative analyses, conserved alphaviral host / pathogen interactions were identified. Approximately 100 unique host proteins were identified as a result of these analyses. Ontological assessments reveal enriched Molecular Functions and Biological Processes relevant to alphaviral infection. Specifically, as anticipated, Poly(A) RNA Binding proteins are significantly enriched in virus specific CLAMP data sets. Moreover, host proteins involved in the regulation of mRNA stability, proteasome mediated degradation, and a number of 14-3-3 proteins were identified. Importantly, these data expand the understanding of alphaviral host / pathogen interactions by identifying conserved interactants.
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Alphavirus/genética , Alphavirus/patogenicidad , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/fisiología , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Alphavirus/fisiología , Animales , Línea Celular , Virus Chikungunya/genética , Virus Chikungunya/patogenicidad , Virus Chikungunya/fisiología , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/patogenicidad , Virus de la Encefalitis Equina Venezolana/fisiología , Evolución Molecular , Células HEK293 , Humanos , Mapas de Interacción de Proteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas/aislamiento & purificación , Ribonucleoproteínas/metabolismo , Virus Sindbis/genética , Virus Sindbis/patogenicidad , Virus Sindbis/fisiología , Especificidad de la EspecieRESUMEN
Alphaviruses are transmitted by an arthropod vector to a vertebrate host. The disease pathologies, cellular environments, immune responses, and host factors are very different in these organisms. Yet, the virus is able to infect, replicate, and assemble into new particles in these two animals using one set of genetic instructions. The balance between conserved mechanisms and unique strategies during virus assembly is critical for fitness of the virus. In this review, we discuss new findings in receptor binding, polyprotein topology, nucleocapsid core formation, and particle budding that have emerged in the last five years and share opinions on how these new findings might answer some questions regarding alphavirus structure and assembly.
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Alphavirus/química , Alphavirus/fisiología , Ensamble de Virus , Alphavirus/patogenicidad , Animales , Artrópodos/virología , Unión Proteica , Proteínas del Envoltorio Viral/metabolismo , Liberación del VirusRESUMEN
Alphaviruses, members of the enveloped, positive-sense, single-stranded RNA Togaviridae family, represent a reemerging public health threat as mosquito vectors expand into new geographic territories. The Old World alphaviruses, which include chikungunya virus, Ross River virus, and Sindbis virus, tend to cause a clinical syndrome characterized by fever, rash, and arthritis, whereas the New World alphaviruses, which consist of Venezuelan equine encephalitis virus, eastern equine encephalitis virus, and western equine encephalitis virus, induce encephalomyelitis. Following recovery from the acute phase of infection, many patients are left with debilitating persistent joint and neurological complications that can last for years. Clues from human cases and studies using animal models strongly suggest that much of the disease and pathology induced by alphavirus infection, particularly atypical and chronic manifestations, is mediated by the immune system rather than directly by the virus. This review discusses the current understanding of the immunopathogenesis of the arthritogenic and neurotropic alphaviruses accumulated through both natural infection of humans and experimental infection of animals, particularly mice. As treatment following alphavirus infection is currently limited to supportive care, understanding the contribution of the immune system to the disease process is critical to developing safe and effective therapies.
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Infecciones por Alphavirus , Alphavirus , Virus de la Encefalitis Equina Venezolana , Alphavirus/genética , Alphavirus/patogenicidad , Infecciones por Alphavirus/inmunología , Infecciones por Alphavirus/patología , Animales , Virus de la Encefalitis Equina Venezolana/genética , Caballos , Humanos , RatonesRESUMEN
Alphaviruses such as Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) are arboviruses that can cause severe zoonotic disease in humans. Both VEEV and EEEV are highly infectious when aerosolized and can be used as biological weapons. Vaccines and therapeutics are urgently needed, but efficacy determination requires animal models. The cynomolgus macaque (Macaca fascicularis) provides a relevant model of human disease, but questions remain whether vaccines or therapeutics can mitigate CNS infection or disease in this model. The documentation of alphavirus encephalitis in animals relies on traditional physiological biomarkers and behavioral/neurological observations by veterinary staff; quantitative measurements such as electroencephalography (EEG) and intracranial pressure (ICP) can recapitulate underlying encephalitic processes. We detail a telemetry implantation method suitable for continuous monitoring of both EEG and ICP in awake macaques, as well as methods for collection and analysis of such data. We sought to evaluate whether changes in EEG/ICP suggestive of CNS penetration by virus would be seen after aerosol exposure of naïve macaques to VEEV IC INH9813 or EEEV V105 strains compared to mock-infection in a cohort of twelve adult cynomolgus macaques. Data collection ran continuously from at least four days preceding aerosol exposure and up to 50 days thereafter. EEG signals were processed into frequency spectrum bands (delta: [0.4 - 4Hz); theta: [4 - 8Hz); alpha: [8-12Hz); beta: [12-30] Hz) and assessed for viral encephalitis-associated changes against robust background circadian variation while ICP data was assessed for signal fidelity, circadian variability, and for meaningful differences during encephalitis. Results indicated differences in delta, alpha, and beta band magnitude in infected macaques, disrupted circadian rhythm, and proportional increases in ICP in response to alphavirus infection. This novel enhancement of the cynomolgus macaque model offers utility for timely determination of onset, severity, and resolution of encephalitic disease and for the evaluation of vaccine and therapeutic candidates.
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Infecciones por Alphavirus/patología , Encéfalo/fisiología , Encefalitis Viral/patología , Presión Intracraneal/fisiología , Alphavirus/aislamiento & purificación , Alphavirus/patogenicidad , Infecciones por Alphavirus/metabolismo , Animales , Biomarcadores/metabolismo , Ritmo Circadiano , Modelos Animales de Enfermedad , Electroencefalografía/métodos , Encefalitis Viral/metabolismo , Femenino , Macaca , Masculino , Índice de Severidad de la Enfermedad , TelemetríaRESUMEN
BACKGROUND: Pancreas disease (PD) is a contagious disease caused by salmonid alphavirus (SAV) with significant economic and welfare impacts on salmon farming. Previous work has shown that higher resistance against PD has underlying additive genetic components and can potentially be improved through selective breeding. To better understand the genetic basis of PD resistance in Atlantic salmon, we challenged 4506 smolts from 296 families of the SalmoBreed strain. Fish were challenged through intraperitoneal injection with the most virulent form of the virus found in Norway (i.e., SAV3). Mortalities were recorded, and more than 900 fish were further genotyped on a 55 K SNP array. RESULTS: The estimated heritability for PD resistance was 0.41 ± 0.017. The genetic markers on two chromosomes, ssa03 and ssa07, showed significant associations with higher disease resistance. Collectively, markers on these two QTL regions explained about 60% of the additive genetic variance. We also sequenced and compared the cardiac transcriptomics of moribund fish and animals that survived the challenge with a focus on candidate genes within the chromosomal segments harbouring QTL. Approximately 200 genes, within the QTL regions, were found to be differentially expressed. Of particular interest, we identified various components of immunoglobulin-heavy-chain locus B (IGH-B) on ssa03 and immunoglobulin-light-chain on ssa07 with markedly higher levels of transcription in the resistant animals. These genes are closely linked to the most strongly QTL associated SNPs, making them likely candidates for further investigation. CONCLUSIONS: The findings presented here provide supporting evidence that breeding is an efficient tool for increasing PD resistance in Atlantic salmon populations. The estimated heritability is one of the largest reported for any disease resistance in this species, where the majority of the genetic variation is explained by two major QTL. The transcriptomic analysis has revealed the activation of essential components of the innate and the adaptive immune responses following infection with SAV3. Furthermore, the complementation of the genomic with the transcriptomic data has highlighted the possible critical role of the immunoglobulin loci in combating PD virus.