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1.
Methods Mol Biol ; 2244: 19-38, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555580

RESUMO

Human cytomegalovirus is routinely isolated by inoculating fibroblast cultures with clinical specimens suspected of harboring HCMV and then monitoring the cultures for cytopathic effects characteristic of this virus. Initially, such clinical isolates are usually strictly cell-associated, but continued propagation in cell culture increases the capacity of an HCMV isolate to release cell-free infectious progeny. Once cell-free infection is possible, genetically homogenous virus strains can be purified by limiting dilution infections. HCMV strains can differ greatly with regard to the titers that can be achieved, the tropism for certain cell types, and the degree to which nonessential genes have been lost during propagation. As there is no ideal HCMV strain for all purposes, the choice of the most appropriate strain depends on the requirements of the particular experiment or project. In this chapter, we provide information that can serve as a basis for deciding which strain may be the most appropriate for a given experiment.


Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/genética , Tropismo Viral/genética , Citomegalovirus/classificação , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/virologia , Fibroblastos/citologia , Humanos , Projetos de Pesquisa , Tropismo Viral/fisiologia , Replicação Viral
2.
Methods Mol Biol ; 2244: 51-81, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555582

RESUMO

The extensive tropism of human cytomegalovirus (HCMV) results in the productive infection of multiple cell types within the human host. However, infection of other cell types, such as undifferentiated cells of the myeloid lineage, give rise to nonpermissive infections. This aspect has been used experimentally to model latent infection, which is known to be established in the pluripotent CD34+ hematopoietic progenitor cell population resident in the bone marrow in vivo. The absence of a tractable animal model for studies of HCMV has resulted in a number of laboratories employing experimental infection of cells in vitro to simulate both HCMV lytic and latent infection. Herein, we will focus on the techniques used in our laboratory for the isolation and use of primary cells to study aspects of HCMV latency, reactivation, and lytic infection.


Assuntos
Citomegalovirus/metabolismo , Cultura Primária de Células/métodos , Antígenos CD34/metabolismo , Diferenciação Celular , Infecções por Citomegalovirus/virologia , Células-Tronco Hematopoéticas/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Tropismo Viral/genética , Tropismo Viral/fisiologia , Ativação Viral , Latência Viral
3.
Methods Mol Biol ; 2244: 83-101, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555583

RESUMO

Of the many research challenges posed by the study of human cytomegalovirus (HCMV) latency, one of the most notable is the requirement for the use of primary hematopoietic cell culture. Culturing hematopoietic progenitor subpopulations requires that consideration be given to maintaining their physiological relevance. We describe a long-standing primary CD34+ hematopoietic progenitor cell (HPC) system as an in vitro model to study HCMV latent infection. Key aspects of the model include infection of primary human CD34+ HPCs prior to ex vivo expansion, a long-term culture with a stromal cell support designed to maintain the ability of stem cells to support hematopoietic reconstitution, and an assay to quantify infectious centers produced prior to and following a reactivation stimulus. Importantly, this system has been used to identify a number of viral determinants of latency or reactivation and findings have been recapitulated in vivo using a humanized mouse model for HCMV latency. Therefore, this system offers a powerful approach to defining virus-host interactions and mechanisms important for HCMV latency and reactivation.


Assuntos
Citomegalovirus/metabolismo , Cultura Primária de Células/métodos , Latência Viral/fisiologia , Antígenos CD34/metabolismo , Diferenciação Celular , Infecções por Citomegalovirus/virologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Transdução de Sinais , Proteínas Virais , Tropismo Viral/genética , Tropismo Viral/fisiologia , Ativação Viral/genética , Ativação Viral/fisiologia
4.
Viruses ; 13(1)2021 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-33477379

RESUMO

Porcine deltacoronavirus (PDCoV) strain OH-FD22 infects poultry and shares high nucleotide identity with sparrow-origin deltacoronaviruses (SpDCoV) ISU73347 and HKU17 strains. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from these SpDCoVs would alter the host and tissue tropism of PDCoV. First, an infectious cDNA clone of PDCoV OH-FD22 strain (icPDCoV) was generated and used to construct chimeric icPDCoVs harboring the S protein of HKU17 (icPDCoV-SHKU17) or the RBD of ISU73347 (icPDCoV-RBDISU). To evaluate their pathogenesis, neonatal gnotobiotic pigs were inoculated orally/oronasally with the recombinant viruses or PDCoV OH-FD22. All pigs inoculated with icPDCoV or OH-FD22 developed severe diarrhea and shed viral RNA at moderate-high levels (7.62-10.56 log10 copies/mL) in feces, and low-moderate levels in nasal swabs (4.92-8.48 log10 copies/mL). No pigs in the icPDCoV-SHKU17 and icPDCoV-RBDISU groups showed clinical signs. Interestingly, low-moderate levels (5.07-7.06 log10 copies/mL) of nasal but not fecal viral RNA shedding were detected transiently at 1-4 days post-inoculation in 40% (2/5) of icPDCoV-SHKU17- and 50% (1/2) of icPDCoV-RBDISU-inoculated pigs. These results confirm that PDCoV infected both the upper respiratory and intestinal tracts of pigs. The chimeric viruses displayed an attenuated phenotype with the loss of tropism for the pig intestine. The SpDCoV S protein and RBD reduced viral replication in pigs, suggesting limited potential for cross-species spillover upon initial passage.


Assuntos
Infecções por Coronavirus/patologia , Intestinos/patologia , Sistema Respiratório/patologia , Glicoproteína da Espícula de Coronavírus/genética , Tropismo Viral/genética , Motivos de Aminoácidos , Animais , Doenças das Aves/virologia , Linhagem Celular , Intestinos/virologia , Proteínas Recombinantes/genética , Sistema Respiratório/virologia , Pardais , Suínos , Doenças dos Suínos/virologia , Virulência/genética
5.
PLoS Genet ; 16(12): e1009272, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332358

RESUMO

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Recombinação Genética/genética , Glicoproteína da Espícula de Coronavírus/genética , Troca Genética/genética , Genes Virais/genética , Genoma Viral/genética , Especificidade de Hospedeiro/genética , Modelos Genéticos , Polimorfismo Genético , /genética , Tropismo Viral/genética
6.
Viruses ; 13(1)2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33375604

RESUMO

Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-ß treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.


Assuntos
Edição de Genes , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Tropismo Viral/genética , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/genética , Humanos , Células Jurkat , RNA Guia , Linfócitos T/imunologia
7.
Cell Rep ; 32(12): 108175, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32946807

RESUMO

To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell transcriptomics across various healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Intestinal goblet cells, enterocytes, and kidney proximal tubule cells appear highly permissive to SARS-CoV-2, consistent with clinical data. Our analysis also predicts non-canonical entry paths for lung and brain infections. Spermatogonial cells and prostate endocrine cells also appear to be permissive to SARS-CoV-2 infection, suggesting male-specific vulnerabilities. Both pro- and anti-viral factors are highly expressed within the nasal epithelium, with potential age-dependent variation, predicting an important battleground for coronavirus infection. Our analysis also suggests that early embryonic and placental development are at moderate risk of infection. Lastly, SCARF expression appears broadly conserved across a subset of primate organs examined. Our study establishes a resource for investigations of coronavirus biology and pathology.


Assuntos
Infecções por Coronavirus/patologia , Mucosa Nasal/metabolismo , Pneumonia Viral/patologia , Receptores Virais/genética , Tropismo Viral/genética , Internalização do Vírus , Células A549 , Animais , Betacoronavirus/crescimento & desenvolvimento , Linhagem Celular , Chlorocebus aethiops , Enterócitos/metabolismo , Perfilação da Expressão Gênica , Células Caliciformes/metabolismo , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única , Células Vero
9.
Acta Virol ; 64(2): 117-130, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32551781

RESUMO

Viruses have evolved sophisticated strategies to subvert immunity to benefit overall viral fitness. Human cytomegalovirus (HCMV, ß-herpesvirus) represents a paradigm of very effective hijacking of gene functions that imitate host encoded immunomodulatory proteins. This co-evolution with the host immune system allowed for establishment of lifelong persistence. The HCMV infection is largely asymptomatic in healthy persons; however, it can induce serious disease in immunocompromised individuals. For this reason, great attention is paid to the development of therapeutics based on HCMV immunomodulatory 'tricks' as well as to the search for active vaccine against HCMV. While comparing the HCMV clinical isolates with extensively passaged laboratory strains, the unique long (UL) b' locus was commonly found to be deleted in HCMV genome while adapted to replication in human fibroblasts in vitro. This missing region, called UL/b' region, encodes up to 22 canonical genes with different functions, as of targeting cellular tropism (e.g. UL133-UL138); viral entry and assembly (e.g. UL128, UL130, UL131A); regulation of immunological synapses (e.g. UL135); inhibition of NK and T cell function (e.g. UL141, UL142, UL148, UL144), ablating activity (e.g. UL146, UL147), but mainly aimed at manipulating the host immune response. Moreover, the presence of UL/b' genomic region dramatically correlates with adverse effects in vaccinated persons, indicating that viral genes in this region play a significant role in controlling virulence. Here, we review how HCMV shapes our immunity by hijacked genes originated from UL/b' locus, discuss their impact in immunomodulation mechanism and how this knowledge may translate to clinical applications. Keywords: immunomodulation; HCMV genes; UL/b' locus; NK cell function; HCMV vaccine; immunity; immunotherapeutics.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus , Genes Virais , Deleção de Sequência , Citomegalovirus/genética , Citomegalovirus/imunologia , Humanos , Glicoproteínas de Membrana/genética , Proteínas Virais/genética , Tropismo Viral/genética , Internalização do Vírus
10.
Viruses ; 12(5)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32414076

RESUMO

Bovine coronavirus (BCoV) is widespread in cattle and wild ruminant populations throughout the world. The virus causes neonatal calf diarrhea and winter dysentery in adult cattle, as well as upper and lower respiratory tract infection in young cattle. We isolated and deep sequenced whole genomes of BCoV from calves with respiratory distress in the south-west of France and conducted a comparative genome analysis using globally collected BCoV sequences to provide insights into the genomic characteristics, evolutionary origins, and global diversity of BCoV. Molecular clock analyses allowed us to estimate that the BCoV ancestor emerged in the 1940s, and that two geographically distinct lineages diverged from the 1960s-1970s. A recombination event in the spike gene (breakpoint at nt 1100) may be at the origin of the genetic divergence sixty years ago. Little evidence of genetic mixing between the spatially segregated lineages was found, suggesting that BCoV genetic diversity is a result of a global transmission pathway that occurred during the last century. However, we found variation in evolution rates between the European and non-European lineages indicating differences in virus ecology.


Assuntos
Doenças dos Bovinos/epidemiologia , Infecções por Coronavirus/epidemiologia , Coronavirus Bovino/genética , Gastroenteropatias/epidemiologia , Gastroenteropatias/veterinária , Infecções Respiratórias/epidemiologia , Animais , Bovinos , Doenças dos Bovinos/transmissão , Infecções por Coronavirus/transmissão , Coronavirus Bovino/patogenicidade , Evolução Molecular , França/epidemiologia , Genoma Viral/genética , Geografia , Filogenia , Infecções Respiratórias/transmissão , Infecções Respiratórias/veterinária , Seleção Genética/genética , Tropismo Viral/genética
11.
Viruses ; 12(5)2020 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-32370153

RESUMO

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a lethal zoonotic pathogen circulating in the Arabian Peninsula since 2012. There is no vaccine for MERS and anti-viral treatment is generally not applicable. We investigated the evolution of the MERS-CoV spike gene sequences and changes in viral loads over time from patients in Saudi Arabia from 2105-2017. All the MERS-CoV strains belonged to lineage 5, and showed high sequence homology (99.9%) to 2017 strains. Recombination analysis showed a potential recombination event in study strains from patients in Saudi Arabia. The spike gene showed eight amino acid substitutions, especially between the A1 and B5 lineage, and contained positively selected codon 1020. We also determined that the viral loads were significantly (p < 0.001) higher in fatal cases, and virus shedding was prolonged in some fatal cases beyond 21 days. The viral concentration peaked during the first week of illness, and the lower respiratory specimens had higher levels of MERS-CoV RNA. The presence of the diversifying selection and the topologies with the structural mapping of residues under purifying selection suggested that codon 1020 might have a role in the evolution of spike gene during the divergence of different lineages. This study will im-prove our understanding of the evolution of MERS-CoV, and also highlights the need for enhanced surveillance in humans and dromedaries. The presence of amino acid changes at the N-terminal domain and structural mapping of residues under positive selection at heptad repeat 1 provides better insight into the adaptive evolution of the spike gene and might have a potential role in virus-host tropism and pathogenesis.


Assuntos
Substituição de Aminoácidos/genética , Infecções por Coronavirus/patologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Camelus/virologia , Dipeptidil Peptidase 4/metabolismo , Evolução Molecular , Feminino , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/genética , RNA Viral/genética , Receptores Virais/genética , Recombinação Genética/genética , Arábia Saudita , Análise de Sequência de RNA , Homologia de Sequência , Carga Viral , Tropismo Viral/genética
12.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32295903

RESUMO

Cell entry by HIV-1 is mediated by its principal receptor, CD4, and a coreceptor, either CCR5 or CXCR4, with viral envelope glycoprotein gp120. Generally, CCR5-using HIV-1 variants, called R5, predominate over most of the course of infection, while CXCR4-using HIV-1 variants (variants that utilize both CCR5 and CXCR4 [R5X4, or dual] or CXCR4 alone [X4]) emerge at late-stage infection in half of HIV-1-infected individuals and are associated with disease progression. Although X4 variants also appear during acute-phase infection in some cases, these variants apparently fall to undetectable levels thereafter. In this study, replication-competent X4 variants were isolated from plasma of drug treatment-naive individuals infected with HIV-1 strain CRF01_AE, which dominantly carries viral RNA (vRNA) of R5 variants. Next-generation sequencing (NGS) confirmed that sequences of X4 variants were indeed present in plasma vRNA from these individuals as a minor population. On the other hand, in one individual with a mixed infection in which X4 variants were dominant, only R5 replication-competent variants were isolated from plasma. These results indicate the existence of replication-competent variants with different coreceptor usage as minor populations.IMPORTANCE The coreceptor switch of HIV-1 from R5 to CXCR4-using variants (R5X4 or X4) has been observed in about half of HIV-1-infected individuals at late-stage infection with loss of CD4 cell count and disease progression. However, the mechanisms that underlie the emergence of CXCR4-using variants at this stage are unclear. In the present study, CXCR4-using X4 variants were isolated from plasma samples of HIV-1-infected individuals that dominantly carried vRNA of R5 variants. The sequences of the X4 variants were detected as a minor population using next-generation sequencing. Taken together, CXCR4-using variants at late-stage infection are likely to emerge when replication-competent CXCR4-using variants are maintained as a minor population during the course of infection. The present study may support the hypothesis that R5-to-X4 switching is mediated by the expansion of preexisting X4 variants in some cases.


Assuntos
Infecções por HIV/imunologia , HIV-1/genética , Receptores CCR5/genética , Receptores CXCR4/genética , Receptores de HIV/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Contagem de Linfócito CD4 , Coinfecção , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Ligação Proteica , RNA Viral/genética , RNA Viral/imunologia , Receptores CCR5/imunologia , Receptores CXCR4/imunologia , Receptores de HIV/genética , Tropismo Viral/genética , Tropismo Viral/imunologia , Ligação Viral , Internalização do Vírus
13.
J Virol ; 94(4)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31776276

RESUMO

Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, as well as sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to the growth of human H1N1 and H3N2 viruses and low-pathogenic avian influenza (LPAI) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) H5N1 virus successfully propagated in HeLa cells to levels comparable to those in a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 virus growth, suggesting the absence of a host factor(s) required for the replication of H1N1, but not H5N1, viruses in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, as well as deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from an H5N1 virus was necessary and sufficient to enable H1N1 virus growth. Overall, this study suggests that the absence of one or more cellular factors in HeLa cells results in abortive replication of H1N1, H3N2, and LPAI viruses, which can be circumvented upon the introduction of H5N1 virus NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into the virus-host interactions that underlie IAV pathogenesis and tropism.IMPORTANCE Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk that these zoonotic viruses may acquire adaptive mutations enabling them to propagate efficiently and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested the growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon the introduction of H5N1 virus HA and NP. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 virus pathogenesis and tropism.


Assuntos
Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Tropismo Viral/genética , Células A549 , Animais , Aves , Linhagem Celular , Cães , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Influenza Aviária/genética , Influenza Aviária/metabolismo , Influenza Humana/genética , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Tropismo Viral/imunologia , Replicação Viral/genética
14.
J Virol ; 94(2)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694947

RESUMO

Infectious bronchitis virus (IBV) infects ciliated epithelial cells in the chicken respiratory tract. While some IBV strains replicate locally, others can disseminate to various organs, including the kidney. Here, we elucidate the determinants for kidney tropism by studying interactions between the receptor-binding domain (RBD) of the viral attachment protein spike from two IBV strains with different tropisms. Recombinantly produced RBDs from the nephropathogenic IBV strain QX and from the nonnephropathogenic strain M41 bound to the epithelial cells of the trachea. In contrast, only QX-RBD binds more extensively to cells of the digestive tract, urogenital tract, and kidneys. While removal of sialic acids from tissues prevented binding of all proteins to all tissues, binding of QX-RBD to trachea and kidney could not be blocked by preincubation with synthetic alpha-2,3-linked sialic acids. The lack of binding of QX-RBD to a previously identified IBV-M41 receptor was confirmed by enzyme-linked immunosorbent assay (ELISA), demonstrating that tissue binding of QX-RBD is dependent on a different sialylated glycan receptor. Using chimeric RBD proteins, we discovered that the region encompassing amino acids 99 to 159 of QX-RBD was required to establish kidney binding. In particular, QX-RBD amino acids 110 to 112 (KIP) were sufficient to render IBV-M41 with the ability to bind to kidney, while the reciprocal mutations in IBV-QX abolished kidney binding completely. Structural analysis of both RBDs suggests that the receptor-binding site for QX is located at a different location on the spike than that of M41.IMPORTANCE Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic (example, IBV-M41) and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field.


Assuntos
Vírus da Bronquite Infecciosa/fisiologia , Rim/virologia , Mutação de Sentido Incorreto , Mucosa Respiratória/virologia , Glicoproteína da Espícula de Coronavírus , Tropismo Viral/genética , Replicação Viral/genética , Substituição de Aminoácidos , Animais , Galinhas/virologia , Células HEK293 , Humanos , Rim/metabolismo , Rim/patologia , Domínios Proteicos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
15.
FEBS Lett ; 593(24): 3583-3608, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31769017

RESUMO

Human adenovirus commonly causes infections of respiratory, gastrointestinal, genitourinary, and ocular surface mucosae. Although most adenovirus eye infections are mild and self-limited, specific viruses within human adenovirus species D are associated with epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular surface infection, which can lead to chronic and/or recurrent, visually disabling keratitis. In this review, we discuss the links between adenovirus ontogeny, genomics, immune responses, and corneal pathogenesis, for those viruses that cause EKC.


Assuntos
Adenovírus Humanos/patogenicidade , Evolução Biológica , Proteínas do Olho/genética , Interações Hospedeiro-Patógeno/genética , Ceratite/genética , Ceratoconjuntivite/genética , Proteínas Virais/genética , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Animais , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Túnica Conjuntiva/virologia , Córnea/imunologia , Córnea/metabolismo , Córnea/patologia , Córnea/virologia , Modelos Animais de Doenças , Proteínas do Olho/imunologia , Regulação da Expressão Gênica , Genômica/métodos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Ceratite/imunologia , Ceratite/patologia , Ceratite/virologia , Ceratoconjuntivite/imunologia , Ceratoconjuntivite/patologia , Ceratoconjuntivite/virologia , Filogenia , Proteínas Virais/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia
16.
PLoS Pathog ; 15(10): e1008057, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31671153

RESUMO

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.


Assuntos
Infecções por Astroviridae/imunologia , Infecções por Astroviridae/veterinária , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Mamastrovirus/fisiologia , Tropismo Viral/genética , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Enterócitos/virologia , Gastroenterite/virologia , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/virologia , Intestino Delgado/citologia , Intestino Delgado/virologia , Mamastrovirus/genética , Mamastrovirus/imunologia , Células Vero , Tropismo Viral/imunologia
17.
J Virol ; 94(1)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597765

RESUMO

Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I, and K582I (K58I in the HA2 subunit), which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1 virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells.IMPORTANCE Receptor specificity of the HA of IAVs is known to be a critical determinant of viral cell tropism. Here, we show that fusion properties of the HA may also play a key role in the tropism. Thus, we demonstrate that IAVs having a relatively low pH optimum of fusion cannot efficiently infect human endothelial cells owing to their relatively high endosomal pH and increased expression of fusion-inhibiting IFITM3 protein. These restrictions can be overcome by IAVs with elevated pH of fusion, such as zoonotic H5N1 and H7N9. Our results illustrate that the infectivity of IAVs depends on an interplay between HA conformational stability, endosomal acidification and IFITM3 expression in target cells, and the extracellular pH. Given significant variation of levels of HA stability among animal, human, and zoonotic IAVs, our findings prompt further studies on the fusion-dependent tropism of IAVs to different cell types in humans and its role in viral host range and pathogenicity.


Assuntos
Endossomos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Vírus Reordenados/genética , Substituição de Aminoácidos , Animais , Cães , Endossomos/virologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Regulação da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração de Íons de Hidrogênio , Virus da Influenza A Subtipo H5N1/metabolismo , Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Vírus Reordenados/metabolismo , Vírus Reordenados/patogenicidade , Relação Estrutura-Atividade , Tropismo Viral/genética , Replicação Viral
18.
Virus Res ; 274: 197771, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31577935

RESUMO

We compared the phenotypes of three mutant AAV2 viruses containing mutations in arginine amino acids (R585, R588 and R484) previously shown to be involved in AAV2 heparan sulfate binding. The transduction efficiencies of wild type and mutant viruses were determined in the eye, the brain and peripheral organs following subretinal, striatal and intravenous injection, respectively, in mice and rats. We found that each of the three mutants (the single mutant R585A; the double mutant R585, 588A; and the triple mutant R585, 588, 484A) had a unique phenotype compared to wt and each other. R585A was completely defective for transducing peripheral organs via intravenous injection, suggesting that R585A may be useful for targeting peripheral organs by substitution of peptide ligands in the capsid surface. In the brain, all three mutants displayed widespread transduction, with the double mutant R585, 588A displaying the greatest spread and the greatest number of transduced neurons. The double mutant was also extremely efficient for retrograde transport, while the triple mutant was almost completely defective for retrograde transport. This suggested that R484 may be directly involved in interaction with the transport machinery. Finally, the double mutant also displayed improved transduction of the eye compared to wild type and the other mutants.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Parvovirinae/fisiologia , Animais , Transporte Axonal/genética , Proteínas do Capsídeo/metabolismo , Feminino , Masculino , Camundongos , Mutação , Parvovirinae/genética , Parvovirinae/metabolismo , Fenótipo , Ligação Proteica , Ratos , Tropismo Viral/genética
19.
Elife ; 82019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31644426

RESUMO

Numerous challenges have impeded HIV-1 vaccine development. Among these is the lack of a convenient small animal model in which to study antibody elicitation and efficacy. We describe a chimeric Rhabdo-Immunodeficiency virus (RhIV) murine model that recapitulates key features of HIV-1 entry, tropism and antibody sensitivity. RhIVs are based on vesicular stomatitis viruses (VSV), but viral entry is mediated by HIV-1 Env proteins from diverse HIV-1 strains. RhIV infection of transgenic mice expressing human CD4 and CCR5, exclusively on mouse CD4+ cells, at levels mimicking those on human CD4+ T-cells, resulted in acute, resolving viremia and CD4+ T-cell depletion. RhIV infection elicited protective immunity, and antibodies to HIV-1 Env that were primarily non-neutralizing and had modest protective efficacy following passive transfer. The RhIV model enables the convenient in vivo study of HIV-1 Env-receptor interactions, antiviral activity of antibodies and humoral responses against HIV-1 Env, in a genetically manipulatable host.


Assuntos
Anticorpos Antivirais/biossíntese , Linfócitos T CD4-Positivos/imunologia , HIV-1/genética , Vírus Reordenados/genética , Vesiculovirus/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Animais , Especificidade de Anticorpos , Antígenos CD4/genética , Antígenos CD4/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/virologia , Modelos Animais de Doenças , Efeito Fundador , Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Vírus Reordenados/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Vesiculovirus/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
20.
PLoS Pathog ; 15(6): e1007790, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194854

RESUMO

Bourbon virus (BRBV) is an emerging tick-borne RNA virus in the orthomyxoviridae family that was discovered in 2014. Although fatal human cases of BRBV have been described, little is known about its pathogenesis, and no antiviral therapies or vaccines exist. We obtained serum from a fatal case in 2017 and successfully recovered the second human infectious isolate of BRBV. Next-generation sequencing of the St. Louis isolate of BRBV (BRBV-STL) showed >99% nucleotide identity to the original reference isolate. Using BRBV-STL, we developed a small animal model to study BRBV-STL tropism in vivo and evaluated the prophylactic and therapeutic efficacy of the experimental antiviral drug favipiravir against BRBV-induced disease. Infection of Ifnar1-/- mice lacking the type I interferon receptor, but not congenic wild-type animals, resulted in uniformly fatal disease 6 to 10 days after infection. RNA in situ hybridization and viral yield assays demonstrated a broad tropism of BRBV-STL with highest levels detected in liver and spleen. In vitro replication and polymerase activity of BRBV-STL were inhibited by favipiravir. Moreover, administration of favipiravir as a prophylaxis or as post-exposure therapy three days after infection prevented BRBV-STL-induced mortality in immunocompromised Ifnar1-/- mice. These results suggest that favipiravir may be a candidate treatment for humans who become infected with BRBV.


Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Infecções por Orthomyxoviridae/prevenção & controle , Pirazinas/farmacologia , Thogotovirus/imunologia , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/imunologia , Thogotovirus/patogenicidade , Células Vero , Tropismo Viral/efeitos dos fármacos , Tropismo Viral/genética , Tropismo Viral/imunologia
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