RESUMO
Nipah virus (NiV) is a highly pathogenic paramyxovirus. The Syrian hamster model recapitulates key features of human NiV disease and is a critical tool for evaluating antivirals and vaccines. Here we describe longitudinal humoral immune responses in NiV-infected Syrian hamsters. Samples were obtained 1-28 days after infection and analyzed by ELISA, neutralization, and Fc-mediated effector function assays. NiV infection elicited robust antibody responses against the nucleoprotein and attachment glycoprotein. Levels of neutralizing antibodies were modest and only detectable in surviving animals. Fc-mediated effector functions were mostly observed in nucleoprotein-targeting antibodies. Antibody levels and activities positively correlated with challenge dose.
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Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention's Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Betacoronavirus/enzimologia , Ebolavirus/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Betacoronavirus/química , Linhagem Celular , Tolerância a Medicamentos/genética , Ebolavirus/efeitos dos fármacos , Ebolavirus/genética , Humanos , Modelos Moleculares , Mutação , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2 , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
Assuntos
Alanina/análogos & derivados , Antivirais/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Macaca mulatta/virologia , Ribonucleotídeos/uso terapêutico , Monofosfato de Adenosina/análogos & derivados , Alanina/farmacocinética , Alanina/farmacologia , Alanina/uso terapêutico , Sequência de Aminoácidos , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular Tumoral , Ebolavirus/efeitos dos fármacos , Feminino , Células HeLa , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Ribonucleotídeos/farmacocinética , Ribonucleotídeos/farmacologiaRESUMO
Nipah virus (NiV; family Paramyxoviridae, genus Henipavirus) infection can cause severe respiratory and neurological disease in humans. The pathophysiology of disease is not fully understood, and it may vary by presentation and clinical course. In this study, we investigate changes in blood chemistry in NiV-infected Syrian hamsters that survived or succumbed to disease. Increased sodium and magnesium and decreased albumin and lactate levels were detected in animals euthanized with severe clinical disease compared with mock-infected controls. When subjects were grouped by clinical syndrome, additional trends were discernable, highlighting changes associated with either respiratory or neurological disease.
Assuntos
Infecções por Henipavirus/sangue , Magnésio/sangue , Vírus Nipah , Albumina Sérica , Sódio/sangue , Alanina Transaminase/sangue , Animais , Cricetinae , Infecções por Henipavirus/virologia , Ácido Láctico/sangue , MesocricetusRESUMO
Using a recombinant Nipah virus expressing a fluorescent protein (ZsG), we visualized virus tropism in the Syrian hamster model. We found that anatomical localization of fluorescence correlated to clinical signs; signal was primarily visualized in the respiratory tract in animals with acute-onset terminal disease, whereas central nervous system localization was seen in animals that succumbed with delayed disease onset. While polymerase chain reaction (PCR) detection corresponded well to ZsG signal, virus was only isolated from some lung, brain, liver, and kidney samples that were ZsG and/or PCR positive, and only from animals euthanized on or before 15 days post infection.
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Infecções do Sistema Nervoso Central/virologia , Infecções por Henipavirus/virologia , Hibridização in Situ Fluorescente/métodos , Vírus Nipah/fisiologia , Infecções Respiratórias/virologia , Animais , Cricetinae , Mesocricetus , Tropismo ViralRESUMO
The error-prone nature of RNA-dependent RNA polymerases drives the diversity of RNA virus populations. Arising within this diversity is a subset of defective viral genomes that retain replication competency, termed defective interfering (DI) genomes. These defects are caused by aberrant viral polymerase reinitiation on the same viral RNA template (deletion DI species) or the nascent RNA strand (copyback DI species). DI genomes have previously been shown to alter the dynamics of a viral population by interfering with normal virus replication and/or by stimulating the innate immune response. In this study, we investigated the ability of artificially produced DI genomes to inhibit Nipah virus (NiV), a highly pathogenic biosafety level 4 paramyxovirus. High multiplicity of infection passaging of both NiV clinical isolates and recombinant NiV in Vero cells generated an extensive DI population from which individual DIs were identified using next-generation sequencing techniques. Assays were established to generate and purify both naturally occurring and in silico-designed DIs as fully encapsidated, infectious virus-like particles termed defective interfering particles (DIPs). We demonstrate that several of these NiV DIP candidates reduced NiV titers by up to 4 logs in vitro. These data represent a proof-of-principle that a therapeutic application of DIPs to combat NiV infections may be an alternative source of antiviral control for this disease.
Assuntos
Genoma Viral , Vírus Nipah/genética , Vírus Nipah/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Vírus Defeituosos , Mesocricetus , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.
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Antivirais/farmacologia , Infecções por Henipavirus/tratamento farmacológico , Vírus Nipah/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Chlorocebus aethiops , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Células HeLa , Infecções por Henipavirus/virologia , Humanos , Mesocricetus , Vírus Nipah/isolamento & purificação , Lectinas de Plantas/uso terapêutico , Células VeroRESUMO
In the absence of approved vaccines and therapeutics for use in humans, Nipah virus (NiV) continues to cause fatal outbreaks of encephalitis and respiratory disease in Bangladesh and India on a near-annual basis. We determined that a single dose of a lipid nanoparticle nucleoside-modified messenger RNA vaccine encoding the soluble Hendra virus glycoprotein protected up to 70% of Syrian hamsters from lethal NiV challenge, despite animals having suboptimally primed immune responses before challenge. These data provide a foundation from which to optimize future messenger RNA vaccination studies against NiV and other highly pathogenic viruses.
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Vírus Hendra , Infecções por Henipavirus/prevenção & controle , Vírus Nipah/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Cricetinae , Relação Dose-Resposta Imunológica , Glicoproteínas , Mesocricetus , Nucleosídeos , RNA Mensageiro/imunologia , Proteínas Virais , Vacinas de mRNARESUMO
Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3'-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus Nipah/genética , Fosfoproteínas/metabolismo , Elongação da Transcrição Genética , Iniciação da Transcrição Genética , Terminação da Transcrição Genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Vírus Nipah/enzimologia , Paramyxovirinae/enzimologia , Paramyxovirinae/genética , Paramyxovirinae/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Replicação ViralRESUMO
Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.
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Proteínas Cromossômicas não Histona/metabolismo , Infecções por Henipavirus/virologia , Vírus Nipah/enzimologia , Animais , Chlorocebus aethiops , Proteínas Cromossômicas não Histona/genética , Células HeLa , Vírus Hendra/metabolismo , Humanos , Mutação , Vírus Nipah/genética , Vírus Nipah/patogenicidade , RNA Interferente Pequeno , Células Vero , Proteínas da Matriz Viral/metabolismoRESUMO
Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites (Balamuthia mandrillaris and Acanthamoeba), six bacterial pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Mycoplasma pneumoniae, Mycobacterium tuberculosis, and Bartonella), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease.
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Infecções do Sistema Nervoso Central/diagnóstico , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , África Subsaariana , Idoso , Idoso de 80 Anos ou mais , Amebozoários/isolamento & purificação , Bactérias/isolamento & purificação , Infecções do Sistema Nervoso Central/microbiologia , Infecções do Sistema Nervoso Central/parasitologia , Infecções do Sistema Nervoso Central/virologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Padrões de Referência , Sensibilidade e Especificidade , Vírus/isolamento & purificação , Adulto JovemRESUMO
UNLABELLED: Bats are important reservoirs for several viruses, many of which cause lethal infections in humans but have reduced pathogenicity in bats. As the innate immune response is critical for controlling viruses, the nature of this response in bats and how it may differ from that in other mammals are of great interest. Using next-generation transcriptome sequencing (mRNA-seq), we profiled the transcriptional response of Pteropus vampyrus bat kidney (PVK) cells to Newcastle disease virus (NDV), an avian paramyxovirus known to elicit a strong innate immune response in mammalian cells. The Pteropus genus is a known reservoir of Nipah virus (NiV) and Hendra virus (HeV). Analysis of the 200 to 300 regulated genes showed that genes for interferon (IFN) and antiviral pathways are highly upregulated in NDV-infected PVK cells, including genes for beta IFN, RIG-I, MDA5, ISG15, and IRF1. NDV-infected cells also upregulated several genes not previously characterized to be antiviral, such as RND1, SERTAD1, CHAC1, and MORC3. In fact, we show that MORC3 is induced by both IFN and NDV infection in PVK cells but is not induced by either stimulus in human A549 cells. In contrast to NDV infection, HeV and NiV infection of PVK cells failed to induce these innate immune response genes. Likewise, an attenuated response was observed in PVK cells infected with recombinant NDVs expressing the NiV IFN antagonist proteins V and W. This study provides the first global profile of a robust virus-induced innate immune response in bats and indicates that henipavirus IFN antagonist mechanisms are likely active in bat cells. IMPORTANCE: Bats are the reservoir host for many highly pathogenic human viruses, including henipaviruses, lyssaviruses, severe acute respiratory syndrome coronavirus, and filoviruses, and many other viruses have also been isolated from bats. Viral infections are reportedly asymptomatic or heavily attenuated in bat populations. Despite their ecological importance to viral maintenance, research into their immune system and mechanisms for viral control has only recently begun. Nipah virus and Hendra virus are two paramyxoviruses associated with high mortality rates in humans and whose reservoir is the Pteropus genus of bats. Greater knowledge of the innate immune response of P. vampyrus bats to viral infection may elucidate how bats serve as a reservoir for so many viruses.
Assuntos
Quirópteros/imunologia , Reservatórios de Doenças/virologia , Perfilação da Expressão Gênica , Infecções por Henipavirus/imunologia , Imunidade Inata , Interferons/imunologia , Vírus Nipah/imunologia , Animais , Quirópteros/genética , Quirópteros/virologia , Vírus Hendra/genética , Vírus Hendra/imunologia , Vírus Hendra/fisiologia , Infecções por Henipavirus/genética , Infecções por Henipavirus/virologia , Humanos , Evasão da Resposta Imune , Interferons/genética , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/fisiologia , Vírus Nipah/genética , Vírus Nipah/fisiologiaRESUMO
In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.
Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Centers for Disease Control and Prevention, U.S. , Surtos de Doenças , Epidemias , Humanos , Laboratórios , Serra Leoa/epidemiologia , Estados UnidosRESUMO
ABSTRACTNipah virus (NiV) is an emerging zoonotic RNA virus that can cause fatal respiratory and neurological disease in animals and humans. Accurate NiV diagnostics and surveillance tools are crucial for the identification of acute and resolved infections and to improve our understanding of NiV transmission and circulation. Here, we have developed and validated a split NanoLuc luciferase NiV glycoprotein (G) biosensor for detecting antibodies in clinical and animal samples. This assay is performed by simply mixing reagents and measuring luminescence, which depends on the complementation of the split NanoLuc luciferase G biosensor following its binding to antibodies. This anti-NiV-G "mix-and-read" assay was validated using the WHO's first international standard for anti-NiV antibodies and more than 700 serum samples from the NiV-endemic country of Bangladesh. Anti-NiV antibodies from survivors persisted for at least 8 years according to both âºNiV-G mix-and-read and NiV neutralization assays. The âºNiV-G mix-and-read assay sensitivity (98.6%) and specificity (100%) were comparable to anti-NiV IgG ELISA performance but failed to detect anti-NiV antibodies in samples collected less than a week following the appearance of symptoms. Overall, the anti-NiV-G biosensor represents a simple, fast, and reliable tool that could support the expansion of NiV surveillance and retrospective outbreak investigations.
RESUMO
Active Nipah virus encephalitis surveillance identified an encephalitis cluster and sporadic cases in Faridpur, Bangladesh, in January 2010. We identified 16 case-patients; 14 of these patients died. For 1 case-patient, the only known exposure was hugging a deceased patient with a probable case, while another case-patient's exposure involved preparing the same corpse for burial by removing oral secretions and anogenital excreta with a cloth and bare hands. Among 7 persons with confirmed sporadic cases, 6 died, including a physician who had physically examined encephalitis patients without gloves or a mask. Nipah virus-infected patients were more likely than community-based controls to report drinking raw date palm sap and to have had physical contact with an encephalitis patient (29% vs. 4%, matched odds ratio undefined). Efforts to prevent transmission should focus on reducing caregivers' exposure to infected patients' bodily secretions during care and traditional burial practices.
Assuntos
Infecção Hospitalar/transmissão , Surtos de Doenças , Encefalite Viral/transmissão , Infecções por Henipavirus/transmissão , Vírus Nipah , Adolescente , Adulto , Arecaceae , Bangladesh/epidemiologia , Bebidas , Sepultamento , Cadáver , Estudos de Casos e Controles , Criança , Pré-Escolar , Infecção Hospitalar/mortalidade , Infecção Hospitalar/virologia , Encefalite Viral/mortalidade , Encefalite Viral/virologia , Monitoramento Epidemiológico , Feminino , Infecções por Henipavirus/mortalidade , Infecções por Henipavirus/virologia , Humanos , Transmissão de Doença Infecciosa do Paciente para o Profissional , Masculino , Pessoa de Meia-Idade , Médicos , Fatores de Risco , Adulto JovemRESUMO
Nipah (NiV) and Hendra (HeV) viruses comprise the genus Henipavirus and are highly pathogenic paramyxoviruses, which cause fatal encephalitis and respiratory disease in humans. Since their respective initial outbreaks in 1998 and 1994, they have continued to cause sporadic outbreaks resulting in fatal disease. Due to their designation as Biosafety Level 4 pathogens, the level of containment required to work with live henipaviruses is available only to select laboratories around the world. This chapter provides an overview of the molecular virology of NiV and HeV including comparisons to other, well-characterized paramyxoviruses. This chapter also describes the sequence diversity present among the henipaviruses.
Assuntos
Genoma Viral , Vírus Hendra/genética , Vírus Nipah/genética , Proteínas Virais/genética , Animais , Quirópteros/virologia , Encefalite Viral/complicações , Encefalite Viral/virologia , Variação Genética , Tamanho do Genoma , Vírus Hendra/classificação , Vírus Hendra/isolamento & purificação , Infecções por Henipavirus/complicações , Infecções por Henipavirus/virologia , Cavalos/virologia , Humanos , Vírus Nipah/classificação , Vírus Nipah/isolamento & purificação , Filogenia , Genética Reversa , Replicação ViralRESUMO
Spillovers of Nipah virus (NiV) from its pteropid bat reservoir into the human population continue to cause near-annual outbreaks of fatal encephalitis and respiratory disease in Bangladesh and India since its emergence in Malaysia over 20 years ago. The current lack of effective antiviral therapeutics against NiV merits further testing of compound libraries against NiV using rapid quantitative antiviral assays. The development of recombinant henipaviruses expressing reporter fluorescence and/or luminescence proteins has facilitated the screening of such libraries. In this chapter, we provide a basic protocol for both types of reporter viruses. Utilizing these live NiV-based reporter assays requires modest instrumentation and sidesteps the labor-intensive steps associated with traditional cytopathic effect or viral antigen-based assays.
Assuntos
Henipavirus , Humanos , Antivirais/farmacologia , Bioensaio , Efeito Citopatogênico Viral , Surtos de DoençasRESUMO
From its discovery in Malaysia in the late 1990s, the spillover of the Nipah virus from its pteropid reservoir into the human population has resulted in sporadic outbreaks of fatal encephalitis and respiratory disease. In this chapter, we revise a previously described quantitative reverse transcription polymerase chain reaction method, which now utilizes degenerate nucleotides at certain positions in the probe and the reverse primer to accommodate the sequence heterogeneity observed within the Nipah henipavirus species.
Assuntos
Encefalite , Infecções por Henipavirus , Humanos , Infecções por Henipavirus/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Surtos de Doenças , NucleotídeosRESUMO
Remdesivir is a nucleotide prodrug with preclinical efficacy against lethal Nipah virus infection in African green monkeys when administered 1 day post inoculation (dpi) (Lo et al., 2019). Here, we determined whether remdesivir treatment was still effective when treatment administration initiation was delayed until 3 dpi. Three groups of six African green monkeys were inoculated with a lethal dose of Nipah virus, genotype Bangladesh. On 3 dpi, one group received a loading dose of 10 mg/kg remdesivir followed by daily dosing with 5 mg/kg for 11 days, one group received 10 mg/kg on 12 consecutive days, and the remaining group received an equivalent volume of vehicle solution. Remdesivir treatment initiation on 3 dpi provided partial protection from severe Nipah virus disease that was dose dependent, with 67% of animals in the high dose group surviving the challenge. However, remdesivir treatment did not prevent clinical disease, and surviving animals showed histologic lesions in the brain. Thus, early administration seems critical for effective remdesivir treatment during Nipah virus infection.