RESUMO
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC 50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k inact /K I of 6.4 x 10 3 M -1 s -1 . LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance Statement: Chikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.
RESUMO
Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro , but their role in infection and pathogenesis is unknown. To examine the in vivo function of vFcγRs in animal hosts closely related to humans, we identified and characterized vFcγRs encoded by rhesus CMV (RhCMV). We demonstrate that Rh05, Rh152/151 and Rh173 represent the complete set of RhCMV vFcγRs, each displaying functional similarities to their respective HCMV orthologs with respect to antagonizing host FcγR activation in vitro . When RhCMV-naïve rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma viremia levels and anti-RhCMV antibody responses were comparable to wildtype infections. However, the duration of plasma viremia was significantly shortened in immunocompetent, but not in CD4+ T cell-depleted animals. Since vFcγRs were not required for superinfection, we conclude that vFcγRs delay control by virus-specific adaptive immune responses, particularly antibodies, during primary infection.
RESUMO
Passively administered monoclonal antibodies (mAbs) given before or after viral infection can prevent or blunt disease. Here, we examine the efficacy of aerosol mAb delivery to prevent infection and disease in rhesus macaques inoculated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant via intranasal and intratracheal routes. SARS-CoV-2 human mAbs or a human mAb directed to respiratory syncytial virus (RSV) are nebulized and delivered using positive airflow via facemask to sedated macaques pre- and post-infection. Nebulized human mAbs are detectable in nasal, oropharyngeal, and bronchoalveolar lavage (BAL) samples. SARS-CoV-2 mAb treatment significantly reduces levels of SARS-CoV-2 viral RNA and infectious virus in the upper and lower respiratory tracts relative to controls. Reductions in lung and BAL virus levels correspond to reduced BAL inflammatory cytokines and lung pathology. Aerosolized antibody therapy for SARS-CoV-2 could be effective for reducing viral burden and limiting disease severity.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Macaca mulatta , COVID-19/patologia , Aerossóis e Gotículas Respiratórios , Pulmão/patologia , Anticorpos Antivirais , Replicação Viral , Anticorpos MonoclonaisRESUMO
Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.
Assuntos
Infecções por Alphavirus , Alphavirus , Vírus Chikungunya , Animais , Camundongos , Masculino , Macaca mulatta , Viremia , Camundongos Endogâmicos C57BL , Anticorpos AntiviraisRESUMO
Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.
Assuntos
Citomegalovirus , Transdução de Sinais , Animais , Camundongos , Humanos , Citomegalovirus/fisiologia , Latência Viral , Diferenciação Celular , Células-Tronco HematopoéticasRESUMO
Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36â%) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Animais , Humanos , Camundongos , Filogenia , Sequência de Bases , MurinaeRESUMO
Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro . Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence Summary: Congenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.
RESUMO
Infections with Chikungunya virus, a mosquito-borne alphavirus, cause an acute febrile syndrome often followed by chronic arthritis that persists for months to years post-infection. Neutralizing antibodies are the primary immune correlate of protection elicited by infection, and the major goal of vaccinations in development. Using convalescent blood samples collected from both endemic and non-endemic human subjects at multiple timepoints following suspected or confirmed chikungunya infection, we identified antibodies with broad neutralizing properties against other alphaviruses within the Semliki Forest complex. Cross-neutralization generally did not extend to the Venezuelan Equine Encephalitis virus (VEEV) complex, although some subjects had low levels of VEEV-neutralizing antibodies. This suggests that broadly neutralizing antibodies elicited following natural infection are largely complex restricted. In addition to serology, we also performed memory B-cell analysis, finding chikungunya-specific memory B-cells in all subjects in this study as remotely as 24 years post-infection. We functionally assessed the ability of memory B-cell derived antibodies to bind to chikungunya virus, and related Mayaro virus, as well as the highly conserved B domain of the E2 glycoprotein thought to contribute to cross-reactivity between related Old-World alphaviruses. To specifically assess the role of the E2 B domain in cross-neutralization, we depleted Mayaro and Chikungunya virus E2 B domain specific antibodies from convalescent sera, finding E2B depletion significantly decreases Mayaro virus specific cross-neutralizing antibody titers with no significant effect on chikungunya virus neutralization, indicating that the E2 B domain is a key target of cross-neutralizing and potentially cross-protective neutralizing antibodies.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Animais , Humanos , Anticorpos Amplamente Neutralizantes , Anticorpos Antivirais , Anticorpos Neutralizantes , GlicoproteínasRESUMO
Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including ß-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O'nyong'nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4+ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Vacinas Virais , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Febre de Chikungunya/prevenção & controle , Epitopos , Formaldeído , Camundongos , ViremiaRESUMO
Chikungunya virus (CHIKV) has re-emerged as a significant human pathogen in the 21st century, causing periodic, and sometimes widespread, outbreaks over the past 15 years. Although mortality is very rare, a debilitating arthralgia is very common and may persist for months or years. There are no antivirals that are approved for the treatment of CHIKV infection, and current treatment options consist of supportive care only. Herein, we demonstrate the efficacy of a CHIKV-specific antibody in the prophylactic and therapeutic treatment of CHIKV in mouse models of disease. The fully human anti-CHIKV monoclonal Ab SVIR023 demonstrated broad in vitro activity against representative strains from the three major CHIKV clades. Therapeutic treatment with SVIR023 administered 1- or 3-days post-infection resulted in reduced virus in various tissues in a dose- and time-dependent manner. Prophylactic treatment up to 4 weeks prior to virus challenge was also effective in preventing disease in mice. Mice treated with SVIR023 and infected with CHIKV were resistant to secondary challenge and no evidence of antibody enhancement of disease was observed. Treatment with SVIR023 was effective in mouse models of CHIKV infection and disease and further evaluation towards clinical development is warranted.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Animais , Anticorpos Antivirais/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Febre de Chikungunya/tratamento farmacológico , Febre de Chikungunya/prevenção & controle , Vírus de DNA , Modelos Animais de Doenças , Camundongos , RoedoresRESUMO
Chikungunya virus (CHIKV) infection in humans is rarely fatal but is often associated with chronic joint and muscle pain. Chronic CHIKV disease is highly debilitating and is associated with viral persistence. To date, there are no approved vaccines or therapeutics to prevent or treat CHIKV infections once they are established. Current palliative treatments aim to reduce joint inflammation and pain associated with acute and chronic CHIKV disease. Development of novel therapeutics that reduces viral loads should positively impact virus inflammatory disease and improve patient outcomes following CHIKV infection. Therapies that target multiple aspects of CHIKV replication cycle should be developed since the virus is capable of rapidly mutating around any single therapeutic. This review summarizes the current status of small molecule inhibitor development against CHIKV.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Vírus , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/genética , Humanos , Replicação ViralRESUMO
Multiple studies worldwide have confirmed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected in wastewater. However, there is a lack of data directly comparing the wastewater SARS-CoV-2 RNA concentration with the prevalence of coronavirus disease 2019 (COVID-19) in individuals living in sewershed areas. Here, we correlate wastewater SARS-CoV-2 signals with SARS-CoV-2 positivity rates in symptomatic and asymptomatic individuals and compare positivity rates in two underserved communities in Portland, Oregon to those reported in greater Multnomah County. 403 individuals were recruited via two COVID-19 testing sites over a period of 16 weeks. The weekly SARS-CoV-2 positivity rate in our cohort ranged from 0 to 21.7% and trended higher than symptomatic positivity rates reported by Multnomah County (1.9-8.7%). Among the 362 individuals who reported symptom status, 76 were symptomatic and 286 were asymptomatic. COVID-19 was detected in 35 participants: 24 symptomatic, 9 asymptomatic, and 2 of unknown symptomatology. Wastewater testing yielded 0.33-149.9 viral RNA genomic copies/L/person and paralleled community COVID-19 positive test rates. In conclusion, wastewater sampling accurately identified increased SARS-CoV-2 within a community. Importantly, the rate of SARS-CoV-2 positivity in underserved areas is higher than positivity rates within the County as a whole, suggesting a disproportionate burden of SARS-CoV-2 in these communities.
RESUMO
Since the explosive outbreak of Zika virus in Brazil and South/Central America in 2015-2016, the frequency of infections has subsided, but Zika virus remains present in this region as well as other tropical and sub-tropical areas of the globe. The most alarming aspect of Zika virus infection is its association with severe birth defects when infection occurs in pregnant women. Understanding the mechanism of Zika virus pathogenesis, which comprises features unique to Zika virus as well as shared with other teratogenic pathogens, is key to future prophylactic or therapeutic interventions. Nonhuman primate-based research has played a significant role in advancing our knowledge of Zika virus pathogenesis, especially with regard to fetal infection. This review summarizes what we have learned from these models and potential future research directions.
Assuntos
Macaca/virologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia , Animais , Brasil/epidemiologia , América Central/epidemiologia , Modelos Animais de Doenças , Surtos de Doenças , Feminino , Gravidez , Complicações Infecciosas na Gravidez/virologia , Zika virus/patogenicidade , Infecção por Zika virus/virologiaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic has challenged clinical diagnostic operations due to supply shortages and high staffing needs to collect nasopharyngeal (NP) swab samples. Saliva is an easily accessible alternative specimen type to overcome some of these challenges. In this study, we first used paired saliva and NP swab specimens (n = 128) to compare test performance characteristics with three RNA extraction platforms, i.e., Maxwell RSC (Promega), MagNA Pure 96 (Roche), and KingFisher Flex (Thermo Fisher Scientific), together with two PCR chemistries, i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2019-nCoV) Centers for Disease Control and Prevention (CDC) quantitative PCR (qPCR) probe assay (Integrated DNA Technologies) and TagPath COVID-19 combination kit (Thermo Fisher Scientific). This study demonstrated that both saliva and NP swab specimens performed well, with 97% agreement when tested by the CDC qPCR chemistry using Maxwell and MagNA Pure RNA extraction platforms. We then compared 12 weeks of saliva and NP swab testing results using two independent asymptomatic populations, including a community surveillance program using saliva samples only (n = 466) and preoperative screening using NP swab samples only (n = 8,461). The positive detection rates among participants with either saliva or NP swab samples were 1.07% and 1.12%, respectively, which confirms the low pretest probabilities for COVID-19 infections in asymptomatic populations. Notably, there was no increased proportion of low-titer cases (inconclusive results) reported in the asymptomatic groups, compared with the all-comers groups (0.21% and 0.66%, respectively, in the community population and 0.25% and 0.49%, respectively, in the preoperative population); this suggests that low-viral-titer carriers can be found similarly in both groups with saliva or NP swab specimens. In summary, saliva can be considered a good alternative for noninvasive but well-instructed self-collection. IMPORTANCE Our study shows that saliva is a noninvasive respiratory secretion sample type that contains equal or more host materials (RNase P), compared with those contained in the corresponding NP swab specimens, in 103 paired samples. SARS-CoV-2 detection with two RNA extraction platforms, Maxwell and MagNA Pure, with CDC qPCR chemistry showed similar test sensitivities for paired specimens. We then analyzed SARS-CoV-2 detections rates in two independent groups of asymptomatic participants, i.e., a group at a community screening station with supervised saliva collection only (n = 466) and a preoperative screening group (n = 8,461) with NP swabbing only. Similar detection rates of 1.07% for the community group and 1.12% for the preoperative group supported the similar test performances in these groups predicted to have low pretest probabilities of infection. With mindful preparation, saliva can be considered for schools and clinical participants when adequate collection education can be provided and compliance can be established.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virologia , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Pandemias , RNA Viral/análise , Manejo de Espécimes/métodos , Adulto JovemRESUMO
Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.
Assuntos
Vírus Chikungunya , Vírus da Encefalite Equina Venezuelana , Quinolonas , Animais , Antivirais/farmacologia , Vírus da Encefalite Equina Venezuelana/genética , Cavalos , Humanos , Quinolonas/farmacologia , Replicação ViralRESUMO
Viruses have evolved diverse strategies to manipulate cellular signaling pathways in order to promote infection and/or persistence. Human cytomegalovirus (HCMV) possesses a number of unique properties that allow the virus to alter cellular events required for infection of a diverse array of host cell types and long-term persistence. Of specific importance is infection of bone marrow derived and myeloid lineage cells, such as peripheral blood monocytes and CD34+ hematopoietic progenitor cells (HPCs) because of their essential role in dissemination of the virus and for the establishment of latency. Viral induced signaling through the Epidermal Growth Factor Receptor (EGFR) and other receptors such as integrins are key control points for viral-induced cellular changes and productive and latent infection in host organ systems. This review will explore the current understanding of HCMV strategies utilized to hijack cellular signaling pathways, such as EGFR, to promote the wide-spread dissemination and the classic life-long herpesvirus persistence.
RESUMO
A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 µM and viral titer reduction (VTR) of 2.5 log at 10 µM with no observed cytotoxicity (CC50 = 169 µM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 µM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.
Assuntos
Antivirais/farmacologia , Derivados de Benzeno/química , Vírus Chikungunya/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacocinética , Antivirais/uso terapêutico , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Derivados de Benzeno/uso terapêutico , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Febre de Chikungunya/tratamento farmacológico , Di-Hidro-Orotato Desidrogenase , Modelos Animais de Doenças , Feminino , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Relação Estrutura-AtividadeRESUMO
Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFNâºR1-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFNâºR1-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses-chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFNâºR1-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas.
Assuntos
Adenoviridae/genética , Alphavirus/imunologia , Febre de Chikungunya/prevenção & controle , Vírus Chikungunya/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteção Cruzada/imunologia , Modelos Animais de Doenças , Feminino , Engenharia Genética/métodos , Vetores Genéticos/genética , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
BACKGROUND: Serological confirmation of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for understanding the dynamics of the pandemic and determining seroprevalence rates within afflicted communities. Common challenges with SARS-CoV-2 serological assays include poor analytical specificity and sensitivity and lack of a serological standard for quantitative assessment of antibody titers. METHODS: To overcome these obstacles, we developed a quantitative enzyme-linked immunosorbent assay based on an optimized 2-dimensional screening assay that utilizes SARS-CoV-2 receptor binding domain (RBD) of spike protein and SARS-CoV-2 spike S1 subunit. RESULTS: A total of 4 SARS-CoV-2-reactive monoclonal antibodies were evaluated for use as serum standards for calibrating assays performed on different days or by different laboratories. This approach provided quantitative analysis of hospitalized reverse transcription polymerase chain reaction-confirmed COVID-19 cases that in some cases reached >100 µg/mL. The assay demonstrated 72% sensitivity based on time points ranging from 2 to 52 days post-symptom onset, with 100% sensitivity at time points measured ≥13 days post-symptom onset and 100% specificity. CONCLUSIONS: Using these optimized reagents and serological standards, we believe this approach will be useful for sensitive and specific determination of seroconversion rates and quantitatively measuring the durability of antiviral antibody responses following SARS-CoV-2 infection or vaccination.
RESUMO
Human cytomegalovirus (HCMV) is a large double-stranded DNA virus and member of the ß-herpesvirus family. HCMV is ubiquitous in the human population and causes lifelong infections. HCMV infection is associated with high morbidity and mortality in immunocompromised individuals and the virus is a major cause of virus-mediated congenital disease. There have been a number of HCMV entry receptors identified that use one of two viral receptor binding complexes, including the gH/gL/gO complex and the pentamer made up of gH/gL/UL128/UL130/UL131a. Cytomegaloviruses (CMVs) are typically host-restricted requiring the use of species-specific modeling and culture conditions. We use rat CMV (RCMV) to study CMV-accelerated vascular disease and chronic allograft rejection. RCMV encodes homologous versions of the entry complex proteins but their incorporation and copy number per virion are still unknown. In this methods article, we describe a novel approach of HiBiT tagging viral proteins in order to detect and quantify protein incorporation into particles. This method is independent of protein-specific antibodies and can be standardized using a commercially available HiBiT protein standard. Using bacterial artificial chromosome (BAC) recombineering, we have constructed two individual viruses containing a HiBiT tag fused to the C'-terminus of either the UL128 homolog (R129) or the UL130 homolog (R131). Viruses containing these mutations were rescued, purified and analyzed. Our data demonstrate that R129 and R131 are both incorporated into RCMV virions at equimolar ratios relative to genome copy number, supporting this antibody-free approach for quantifying viral protein incorporation and its application toward the identification of domains required for incorporation.