RESUMO
We previously found that infection with human parainfluenza virus type 2 (hPIV-2), a member of the genus Orthorubulavirus, family Paramyxoviridae, causes filamentous actin (F-actin) formation to promote viral growth. In the present study, we investigated whether similar regulation of F-actin formation is observed in infections with other rubulaviruses, such as parainfluenza virus type 5 (PIV-5) and simian virus 41 (SV41). Infection with these viruses caused F-actin formation and RhoA activation, which promoted viral growth. These results indicate that RhoA-induced F-actin formation is important for efficient growth of these rubulaviruses. Only SV41 and hPIV-2 V and P proteins bound to Graf1, while the V and P proteins of PIV-5, mumps virus, and hPIV-4 did not bind to Graf1. In contrast, the V proteins of these rubulaviruses bound to both inactive RhoA and profilin 2. These results suggest that there are common and unique mechanisms involved in regulation of F-actin formation by members of the genus Orthorubulavirus.
Assuntos
Actinas/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Vírus da Parainfluenza 5/metabolismo , Infecções por Rubulavirus/metabolismo , Rubulavirus/metabolismo , Actinas/química , Actinas/genética , Animais , Linhagem Celular , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/crescimento & desenvolvimento , Vírus da Parainfluenza 5/genética , Vírus da Parainfluenza 5/crescimento & desenvolvimento , Ligação Proteica , Rubulavirus/genética , Rubulavirus/crescimento & desenvolvimento , Infecções por Rubulavirus/genética , Infecções por Rubulavirus/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
OBJECTIVES: To examine outbreak response-associated costs, lessons learned, and challenges encountered during a local health department's response to a mumps outbreak. DESIGN: We conducted semistructured interviews with individuals directly involved in the response to a mumps outbreak and analyzed outbreak response-associated cost data. SETTING: In March-July 2018, a mumps outbreak occurred in Chester County, Pennsylvania. The outbreak primarily affected an immigrant community, some of whom spoke little or no English and were uninsured and/or undocumented. This necessitated an urgent response from the Chester County Health Department, which implemented a variety of public health interventions, including outreach to local health care providers and the execution of vaccination clinics at 2 local mushroom farms where case contacts worked. A total of 39 suspected or confirmed mumps cases were reported in Chester County, and 22 suspected or confirmed cases were reported by 2 neighboring jurisdictions. PARTICIPANTS: Health department employees (n = 7) and community partners (n = 2). Areas of expertise included emergency preparedness, nursing, medicine, disease surveillance, and epidemiology. MAIN OUTCOME MEASURE: Operational challenges encountered and lessons learned during the mumps outbreak response, including outbreak response-associated costs, which could inform other communities' planning and preparedness for outbreaks in similar populations and improve outbreak response operations. RESULTS: Immigration status emerged as a key challenge, which highlighted the importance of building trust through community outreach and partnerships and the need for culturally competent communication. In addition, vaccine availability, accessibility, and cost played a major role in response operations and necessitated the involvement of community partners to mitigate these barriers. Outbreak response-associated costs totaled $35 305. CONCLUSIONS: The challenges that occurred in this outbreak are broadly relevant to outbreaks that affect similar immigrant communities. Health departments that serve such populations can utilize these lessons to develop improved outbreak response plans that account for these challenges.
Assuntos
Barreiras de Comunicação , Surtos de Doenças/estatística & dados numéricos , Emigrantes e Imigrantes/estatística & dados numéricos , Caxumba/diagnóstico , Humanos , Caxumba/epidemiologia , Caxumba/transmissão , Pennsylvania/epidemiologia , Saúde Pública/métodos , Saúde Pública/estatística & dados numéricos , Rubulavirus/patogenicidadeRESUMO
Although human rubulavirus 2 (HPIV2) is an important respiratory pathogen, little is known about its molecular epidemiology. We performed a comparative analysis of the full-length genomes of fourteen HPIV2 isolates belonging to different genotypes. Additionally, evolutionary analyses (phylogenetic reconstruction, sequence identity, detection of recombination and adaptive evolution) were conducted. Our study presents a systematic comparative genetic analysis that complements prior analyses and utilizes full-length HPIV2 genomes to provide a basis for future work on the clinical significance, molecular variation and conservation, and evolution of HPIV2.
Assuntos
Infecções por Rubulavirus/virologia , Rubulavirus/genética , Evolução Molecular , Genoma Viral , Genômica , Genótipo , Humanos , Filogenia , Rubulavirus/classificação , Rubulavirus/isolamento & purificaçãoRESUMO
UNLABELLED: We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus type 5 (PIV5), selectively inhibiting the translation of PIV5 mRNAs. Here we report that while PIV2, PIV5, and mumps virus (MuV) are sensitive to IFIT1, nonrubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV), and canine distemper virus (CDV) are resistant. The IFIT1 sensitivity of PIV5 was not rescued by coinfection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. While the translations of PIV2, PIV5, and MuV mRNAs were directly inhibited by IFIT1, the translations of PIV3, SeV, and CDV mRNAs were not. Using purified human mRNA-capping enzymes, we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5' guanosine nucleoside cap (which need not be N7 methylated) and that this sensitivity is partly abrogated by 2'O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2'O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2'-O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed. IMPORTANCE: Paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. Thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. One factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (IFN) system. Here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting IFN-induced antiviral state. Strikingly, all the rubulaviruses tested were sensitive to the antiviral action of ISG56/IFIT1, while all the other paramyxoviruses tested were resistant. We developed novel in vitro biochemical assays to investigate the mechanism of action of IFIT1, demonstrating that the mRNAs of rubulaviruses can be directly inhibited by IFIT1 and that this is at least partially because their mRNAs are not correctly methylated.
Assuntos
Proteínas de Transporte/farmacologia , Paramyxoviridae/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Rubulavirus/genética , Células A549 , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Humanos , Interferon-alfa/metabolismo , Metilação , Vírus da Caxumba/genética , Vírus da Parainfluenza 5/genética , Capuzes de RNA/genética , RNA Viral/genética , Proteínas de Ligação a RNA , Vírus Sendai/genética , Replicação Viral/genéticaRESUMO
Leader sequence, located at the 3' terminus of paramyxovirus genomes, determines the degree of viral transcription and replication. The essential nucleotides in the leader sequence that influence viral propagation, however, have not been investigated in detail. In the present study, we show that polymerase complex of human parainfluenza virus type 2 (hPIV2) uses a luciferase-encoding hPIV2 mini-genome possessing the leader sequence from other closely related viruses as a template. Furthermore, we demonstrate that although hPIV2 polymerase complex can recognize the leader sequence of hPIV4B, mumps virus (MuV) and PIV5 as well as Newcastle disease virus (NDV), it cannot recognize measles virus, hPIV1, Sendai virus (SeV) or hPIV3. We could obtain the chimeric hPIV2 possessing the leader sequence from hPIV4B, MuV and PIV5, but not from other species, including NDV and SeV. These results reveal that although hPIV2 polymerase complex can recognize the leader sequence from rubulaviruses to achieve efficient viral infection, this does not apply to viruses belonging to other genus. A comparison of leader sequence nucleotides among paramyxoviruses highlights the importance of the conservation in the first 13 nucleotides for infectious hPIV2 growth.
Assuntos
Regiões 3' não Traduzidas , Vírus da Parainfluenza 2 Humana/enzimologia , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Rubulavirus/genética , Animais , Linhagem Celular , Humanos , Ligação Proteica , Rubulavirus/fisiologia , Transcrição Gênica , Replicação ViralRESUMO
Tetherin (BST-2/CD317/HM1.24) is an anti-viral factor that restricts the budding of several enveloped viruses. Most of these viruses have evolved to encode tetherin antagonists. Our previous study demonstrated that the growth of human parainfluenza virus type 2 (hPIV-2), a member of the genus Rubulavirus in the family Paramyxoviridae, was inhibited by tetherin, and its V protein decreases the amount of cell surface tetherin by the interaction. In the present study, we investigated whether tetherin inhibits the growth of other rubulaviruses including PIV-5, mumps virus (MuV), simian virus 41, and hPIV-4, and whether their V proteins act as tetherin antagonists. Plaque assay demonstrated that the growth of PIV-5 and MuV was inhibited by tetherin. Flow cytometry and immunoblot analyses revealed that the infection of PIV-5 and MuV caused reduction of cell surface tetherin without affecting total amount of tetherin. Immunoprecipitation analysis showed that all V proteins of rubulaviruses tested bound to tetherin. These results suggest that tetherin antagonism by V proteins is common among the genus Rubulavirus.
Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Rubulavirus/imunologia , Rubulavirus/fisiologia , Proteínas Virais/metabolismo , Animais , Antígenos CD , Células COS , Chlorocebus aethiops , Citometria de Fluxo , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Immunoblotting , Imunoprecipitação , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Células Vero , Ensaio de Placa Viral , Proteínas Virais/genéticaRESUMO
We report the complete genome sequences of four neurovirulent isolates of porcine rubulavirus (PorPV) from 2015 and one historical PorPV isolate from 1984 obtained by next-generation sequencing. A phylogenetic tree constructed using the individual sequences of the complete HN genes of the 2015 isolates and other historical sequences deposited in the GenBank database revealed that several recent neurovirulent isolates of PorPV (2008-2015) cluster together in a separate clade. Phylogenetic analysis of the complete genome sequences revealed that the neurovirulent strains of PorPV that circulated in Mexico during 2015 are genetically different from the PorPV strains that circulated during the 1980s.
Assuntos
Genoma Viral , Filogenia , Infecções por Rubulavirus/veterinária , Rubulavirus/isolamento & purificação , Doenças dos Suínos/virologia , Animais , Sequência de Bases , México , Dados de Sequência Molecular , RNA Viral/genética , Rubulavirus/classificação , Rubulavirus/genética , Infecções por Rubulavirus/virologia , SuínosRESUMO
BACKGROUND: Unlike influenza viruses, little is known about the prevalence and seasonality of other respiratory viruses because laboratory surveillance for non-influenza respiratory viruses is not well developed or supported in China and other resource-limited countries. We studied the interference between seasonal epidemics of influenza viruses and five other common viruses that cause respiratory illnesses in Hong Kong from 2014 to 2017. METHODS: The weekly laboratory-confirmed positive rates of each virus were analyzed from 2014 to 2017 in Hong Kong to describe the epidemiological trends and interference between influenza viruses, respiratory syncytial virus (RSV), parainfluenza virus (PIV), adenovirus, enterovirus and rhinovirus. A sinusoidal model was established to estimate the peak timing of each virus by phase angle parameters. RESULTS: Seasonal features of the influenza viruses, PIV, enterovirus and adenovirus were obvious, whereas annual peaks of RSV and rhinovirus were not observed. The incidence of the influenza viruses usually peaked in February and July, and the summer peaks in July were generally caused by the H3 subtype of influenza A alone. When influenza viruses were active, other viruses tended to have a low level of activity. The peaks of the influenza viruses were not synchronized. An epidemic of rhinovirus tended to shift the subsequent epidemics of the other viruses. CONCLUSION: The evidence from recent surveillance data in Hong Kong suggests that viral interference during the epidemics of influenza viruses and other common respiratory viruses might affect the timing and duration of subsequent epidemics of a certain or several viruses.
Assuntos
Influenza Humana/epidemiologia , Infecções Respiratórias/epidemiologia , Adenoviridae/isolamento & purificação , Pré-Escolar , Enterovirus/isolamento & purificação , Epidemias , Hong Kong/epidemiologia , Humanos , Incidência , Influenza Humana/virologia , Nasofaringe/virologia , Orthomyxoviridae/isolamento & purificação , Faringe/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/virologia , Rhinovirus/isolamento & purificação , Rubulavirus/isolamento & purificação , Estações do AnoRESUMO
Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.
Assuntos
Clonagem Molecular , Expressão Gênica , Proteína HN , Imunogenicidade da Vacina , Rubulavirus , Vacinas Virais , Animais , Escherichia coli , Feminino , Proteína HN/biossíntese , Proteína HN/imunologia , Proteína HN/isolamento & purificação , Proteína HN/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Rubulavirus/enzimologia , Rubulavirus/imunologia , Suínos , Vacinas Virais/biossíntese , Vacinas Virais/imunologiaRESUMO
Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.
Assuntos
Infecções por Rubulavirus/veterinária , Rubulavirus/genética , Doenças dos Suínos/virologia , Animais , DNA Complementar , Surtos de Doenças/veterinária , Genes Virais , Variação Genética , México/epidemiologia , Filogenia , RNA Viral , Rubulavirus/classificação , Infecções por Rubulavirus/epidemiologia , Infecções por Rubulavirus/virologia , Análise de Sequência de RNA , Suínos , Doenças dos Suínos/epidemiologia , Proteínas Virais/genéticaRESUMO
Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be particularly useful to identify the main respiratory viruses directly from clinical samples, after nucleic acid extraction, and, also, to screen a large number of patients for epidemiological studies.
Assuntos
Adenoviridae/isolamento & purificação , Bocavirus Humano/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Vírus de RNA/isolamento & purificação , Infecções Respiratórias/virologia , Adenoviridae/classificação , Coronavirus/classificação , Coronavirus/isolamento & purificação , Enterovirus/classificação , Enterovirus/isolamento & purificação , Bocavirus Humano/classificação , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/classificação , Vírus da Influenza B/isolamento & purificação , Metapneumovirus/classificação , Metapneumovirus/isolamento & purificação , Vírus de RNA/classificação , Vírus Sinciciais Respiratórios/classificação , Vírus Sinciciais Respiratórios/isolamento & purificação , Respirovirus/classificação , Respirovirus/isolamento & purificação , Rubulavirus/classificação , Rubulavirus/isolamento & purificaçãoRESUMO
The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion.
Assuntos
Proteína HN/química , Dobramento de Proteína , Rubulavirus/enzimologia , Animais , Chlorocebus aethiops , Cricetinae , Cristalografia por Raios X , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Estrutura Terciária de Proteína , Rubulavirus/genética , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Internalização do VírusRESUMO
Bats carry a variety of paramyxoviruses that impact human and domestic animal health when spillover occurs. Recent studies have shown a great diversity of paramyxoviruses in an urban-roosting population of straw-colored fruit bats in Ghana. Here, we investigate this further through virus isolation and describe two novel rubulaviruses: Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2). The viruses form a phylogenetic cluster with each other and other bat-derived rubulaviruses, such as Tuhoko viruses, Menangle virus, and Tioman virus. We developed AchPV1- and AchPV2-specific serological assays and found evidence of infection with both viruses in Eidolon helvum across sub-Saharan Africa and on islands in the Gulf of Guinea. Longitudinal sampling of E. helvum indicates virus persistence within fruit bat populations and suggests spread of AchPVs via horizontal transmission. We also detected possible serological evidence of human infection with AchPV2 in Ghana and Tanzania. It is likely that clinically significant zoonotic spillover of chiropteran paramyxoviruses could be missed throughout much of Africa where health surveillance and diagnostics are poor and comorbidities, such as infection with HIV or Plasmodium sp., are common.
Assuntos
Quirópteros/virologia , Infecções por Rubulavirus/veterinária , Infecções por Rubulavirus/virologia , Rubulavirus/classificação , Rubulavirus/isolamento & purificação , Zoonoses/epidemiologia , Adolescente , Adulto , África/epidemiologia , Animais , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Análise por Conglomerados , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Rubulavirus/genética , Rubulavirus/patogenicidade , Infecções por Rubulavirus/epidemiologia , Análise de Sequência de DNA , Estudos SoroepidemiológicosRESUMO
For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.
Assuntos
Proteína HN/metabolismo , Rubulavirus/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Animais , Fusão Celular , Linhagem Celular , Cricetinae , Análise Mutacional de DNA , Proteína HN/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Rubulavirus/genética , Proteínas Virais de Fusão/genéticaRESUMO
Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.
Assuntos
Vírus Defeituosos/genética , Genoma Viral , Infecções por Rubulavirus/imunologia , Infecções por Rubulavirus/virologia , Rubulavirus/genética , Animais , Linhagem Celular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferons/genética , Interferons/imunologia , Infecções por Rubulavirus/genética , Proteínas Virais/genéticaRESUMO
Parainfluenza virus 5 (PIV5) is a promising viral vector for vaccine development. PIV5 is safe, stable, efficacious, cost-effective to produce and, most interestingly, it overcomes preexisting antivector immunity. We have recently reported that PIV5 expressing the hemagglutinin (HA) from highly pathogenic avian influenza (HPAI) virus H5N1 (PIV5-H5) provides sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. It is thought that induction of apoptosis can lead to enhanced antigen presentation. Previously, we have shown that deleting the SH gene and the conserved C terminus of the V gene in PIV5 results in mutant viruses (PIV5ΔSH and PIV5VΔC) that enhance induction of apoptosis. In this study, we inserted the HA gene of H5N1 into PIV5ΔSH (PIV5ΔSH-H5) or PIV5VΔC (PIV5VΔC-H5) and compared their efficacies as vaccine candidates to PIV5-H5. We have found that PIV5ΔSH-H5 induced the highest levels of anti-HA antibodies, the strongest T cell responses, and the best protection against an H5N1 lethal challenge in mice. These results suggest that PIV5ΔSH is a better vaccine vector than wild-type PIV5.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Parainfluenza 5/genética , Rubulavirus/genética , Animais , Anticorpos Antivirais/biossíntese , Apresentação de Antígeno , Apoptose , Chlorocebus aethiops , Feminino , Genes Virais , Vetores Genéticos/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vírus da Parainfluenza 5/imunologia , Rubulavirus/imunologia , Linfócitos T/imunologia , Células VeroRESUMO
Human parainfluenza viruses (HPIV) are important causes of respiratory tract infections in young children. To characterize the molecular epidemiology of an HPIV outbreak occurring in Korea during 2006, genetic analysis of 269 cell culture isolates from HPIV-infected children, was conducted using nested reverse transcription-PCR (RT-PCR). HPIV-1 was detected in 70.3% of tested samples (189/269). The detection rate of HPIV-2 and HPIV-3 was 1.5% (4/269) and 9.3% (25/269), respectively. Mixed HPIV-1, -2 and -3 infections were detected in 19.0% (51/269): HPIV-1 and HPIV-2 in 15, HPIV-1 and HPIV-3 in 26, HPIV-2 and HPIV-3 in 6, and HPIV-1, -2 and -3 in 4. Of these positive samples for three different types HIPV-1, -2, and -3, two each representative strains were selected, the full length of hemagglutinin-neuraminidase (HN) gene for HPIV was amplified by RT-PCR, and sequenced. Multiple alignment analysis, based on reference sequence of HPIV-1, -2, and -3 strains available in GenBank, showed that the identity of nucleotide and deduced amino acid sequences was 92.4-97.6% and 92.7-97.9%, respectively, for HPIV-1, 88.5-99.8% and 88.6-100% for HPIV-2, and 96.3-99.5% and 95.0-99.3% for HPIV-3, respectively. Phylogenetic analysis showed that HPIV-1, -2, and -3 strains identified in this study were closely related among the strains in the same type with no significant genetic variability. These results show that HPIV of multiple imported sources was circulating in Korea.
Assuntos
Infecções por Paramyxoviridae/epidemiologia , Infecções por Paramyxoviridae/virologia , Respirovirus/classificação , Respirovirus/genética , Rubulavirus/classificação , Rubulavirus/genética , Criança , Pré-Escolar , Coinfecção/epidemiologia , Coinfecção/virologia , Variação Genética , Proteína HN/genética , Humanos , Lactente , Epidemiologia Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Prevalência , República da Coreia/epidemiologia , Respirovirus/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rubulavirus/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Fusion is mediated by the viral fusion (F) protein, and it undergoes large irreversible conformational changes to cause membrane merger. The C terminus of PIV5 F contains a membrane-proximal 7-residue external region (MPER), followed by the transmembrane (TM) domain and a 20-residue cytoplasmic tail. To study the sequence requirements of the F protein C terminus for fusion, we constructed chimeras containing the ectodomain of parainfluenza virus 5 F (PIV5 F) and either the MPER, the TM domain, or the cytoplasmic tail of the F proteins of the paramyxoviruses measles virus, mumps virus, Newcastle disease virus, human parainfluenza virus 3, and Nipah virus. The chimeras were expressed, and their ability to cause cell fusion was analyzed. The chimeric proteins were variably expressed at the cell surface. We found that chimeras containing the ectodomain of PIV5 F with the C terminus of other paramyxoviruses were unable to cause cell fusion. Fusion could be restored by decreasing the activation energy of refolding through introduction of a destabilizing mutation (S443P). Replacing individual regions, singly or doubly, in the chimeras with native PIV5 F sequences restored fusion to various degrees, but it did not have an additive effect in restoring activity. Thus, the F protein C terminus may be a specific structure that only functions with its cognate ectodomain. Alanine scanning mutagenesis of MPER indicates that it has a regulatory role in fusion since both hyperfusogenic and hypofusogenic mutations were found.
Assuntos
Paramyxovirinae/química , Paramyxovirinae/genética , Rubulavirus/genética , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Fusão Celular , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mutagênese , Mutação , Infecções por Paramyxoviridae/veterinária , Infecções por Paramyxoviridae/virologia , Paramyxovirinae/fisiologia , Rubulavirus/química , Rubulavirus/fisiologia , Alinhamento de Sequência , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/fisiologiaRESUMO
RIG-I and mda-5 are activated by viral RNA and stimulate type I interferon production. Laboratory of genetics and physiology 2 (LGP2) shares homology with RIG-I and mda-5 but lacks the CARD domains required for signaling. The V proteins of paramyxoviruses limit interferon induction by binding mda-5 and preventing its activation; however, they do not bind RIG-I and have not been considered inhibitors of RIG-I signaling. Here we uncover a novel mechanism of RIG-I inhibition in which the V protein of parainfluenzavirus type 5 (PIV5; formerly known as simian virus type 5 [SV5]) interacts with LGP2 and cooperatively inhibits induction by RIG-I ligands. A complex between RIG-I and LGP2 is observed in the presence of PIV5-V, and we propose that this complex is refractory to activation by RIG-I ligands. The V proteins from other paramyxoviruses also bind LGP2 and demonstrate LGP2-dependent inhibition of RIG-I signaling. This is significant, because it demonstrates a general mechanism for the targeting of the RIG-I pathway by paramyxoviruses.
Assuntos
RNA Helicases DEAD-box/metabolismo , Interferon beta/metabolismo , Interferons/metabolismo , RNA Helicases/metabolismo , Infecções por Rubulavirus/enzimologia , Rubulavirus/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Humanos , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferons/genética , Dados de Sequência Molecular , Ligação Proteica , RNA Helicases/química , RNA Helicases/genética , Receptores Imunológicos , Rubulavirus/genética , Infecções por Rubulavirus/genética , Infecções por Rubulavirus/virologia , Alinhamento de Sequência , Proteínas Virais/genéticaRESUMO
Blue-eye disease is an emergent viral swine infection caused by porcine rubulavirus (PoRV). We have developed a qRT-PCR method to detect and quantify expression of the nucleoprotein gene for different PoRV strains. The limit of detection for this assay was 10(2) copies of synthetic RNA. Viral RNA from PoRV was detectable at a TCID50 of 0.01. Significant differences were observed between viral RNA quantification and virus titration results for nine PoRV strains. For nasal and oral swab samples that were collected from experimentally infected pigs, the qRT-PCR assay was more sensitive (87.1-83.9 %) for the detection of positive samples than methods involving isolation of virus. The implementation of highly sensitive assays that yield results quickly will be of great assistance in the eradication of PoRV from Mexico. We also believe that the newly developed qRT-PCR assay will help reduce the spread of this viral infection to other countries.