RESUMO
Wastewater surveillance is a promising tool for population-level monitoring of the spread of infectious diseases, such as the coronavirus disease 2019 (COVID-19). Different from clinical specimens, viruses in community-scale wastewater samples need to be concentrated before detection because viral RNA is highly diluted. The present study evaluated eleven different virus concentration methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater. First, eight concentration methods of different principles were compared using spiked wastewater at a starting volume of 30 mL. Ultracentrifugation was the most effective method with a viral recovery efficiency of 25 ± 6%. The second-best option, AlCl3 precipitation method, yielded a lower recovery efficiency, only approximately half that of the ultracentrifugation method. Second, the potential of increasing method sensitivity was explored using three concentration methods starting with a larger volume of 1000 mL. Although ultracentrifugation using a large volume outperformed the other two large-volume methods, it only yielded a comparable method sensitivity as the ultracentrifugation using a small volume (30 mL). Thus, ultracentrifugation using less volume of wastewater is more preferable considering the sample processing throughput. Third, a comparison of two viral RNA extraction methods showed that the lysis-buffer-based extraction method resulted in higher viral recovery efficiencies, with cycle threshold (Ct) values 0.9-4.2 lower than those obtained for the acid-guanidinium-phenol-based method using spiked samples. These results were further confirmed by using positive wastewater samples concentrated by ultracentrifugation and extracted separately by the two viral RNA extraction methods. In summary, concentration using ultracentrifugation followed by the lysis buffer-based extraction method enables sensitive and robust detection of SARS-CoV-2 for wastewater surveillance.
Assuntos
COVID-19 , Vírus , Humanos , RNA Viral , SARS-CoV-2 , Vírus/genética , Águas Residuárias , Vigilância Epidemiológica Baseada em Águas ResiduáriasRESUMO
The initial stage of host cell infection by influenza A viruses (IAV) is mediated through interaction of the viral haemagglutinin (HA) with cell surface glycans. The binding requirement of IAVs for Galß(1,4)Glc/ GlcNAc (lactose/lactosamine) glycans with a terminal α(2,6)-linked (human receptors) or α(2,3)-linked (avian receptors) N-acetylneuraminic residue commonly found on N-glycans, is well-established. However the role and significance of sialylated Galß(1,3)GalNAc (core 1) epitopes that are typical O-glycoforms in influenza virus pathogenesis remains poorly detailed. Here we report a multidisciplinary study using NMR spectroscopy, virus neutralization assays and molecular modelling, into the potential for IAV to engage sialyl-Galß(1,3)GalNAc O-glycoforms for cell attachment. H5 containing virus like particles (VLPs) derived from an H5N1 avian IAV strain show a significant involvement of the O-glycan-specific GalNAc residue, coordinated by a EQTKLY motif conserved in highly pathogenic avian influenza (HPAI) strains. Notably, human pandemic H1N1 influenza viruses shift the preference from 'human-like' α(2,6)-linkages in sialylated Galß(1,4)Glc/GlcNAc fragments to 'avian-like' α(2,3)-linkages in sialylated Galß(1,3)GalNAc without involvement of the GalNAc residue. Overall, our study suggests that sialylated Galß(1,3)GalNAc as O-glycan core 1 glycoforms are involved in the influenza A virus life cycle and play a particularly crucial role during infection of HPAI strains.
Assuntos
Vírus da Influenza A/fisiologia , Polissacarídeos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A Subtipo H3N2/fisiologia , Virus da Influenza A Subtipo H5N1/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A/metabolismo , Simulação de Dinâmica Molecular , Polissacarídeos/química , Ligação Proteica , Conformação ProteicaRESUMO
Since their first isolation in 2013, influenza A/H5N6 viruses have spread amongst poultry across multiple provinces in China and to Laos, Vietnam and Myanmar. So far, there have been 14 human H5N6 infections with 10 fatalities.We investigated the tropism, replication competence and cytokine induction of one human and two avian H5N6 isolates in ex vivo and in vitro cultures derived from the human respiratory tract. Virus tropism and replication were studied in ex vivo cultures of human nasopharynx, bronchus and lung. Induction of cytokines and chemokines was measured in vitro in virus-infected primary human alveolar epithelial cells.Human H5N6 virus replicated more efficiently than highly pathogenic avian influenza (HPAI) H5N1 virus and as efficiently as H1N1pdm in ex vivo human bronchus and lung and was also able to replicate in ex vivo cultures of human nasopharynx. Avian H5N6 viruses replicated less efficiently than H1N1pdm in human bronchial tissues and to similar titres as HPAI H5N1 in the lung. While the human H5N6 virus had affinity for avian-like receptors, the two avian isolates had binding affinity for both avian- and human-like receptors. All three H5N6 viruses were less potent inducers of pro-inflammatory cytokines compared with H5N1 virus.Human H5N6 virus appears better adapted to infect the human airways than H5N1 virus and may pose a significant public health threat.
Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Sistema Respiratório/virologia , Tropismo Viral , Replicação Viral , Células Epiteliais Alveolares/virologia , Animais , Aves , Células Cultivadas , Quimiocinas/imunologia , Citocinas/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A/patogenicidade , Influenza Aviária/imunologia , Masculino , Pessoa de Meia-Idade , Sistema Respiratório/patologia , Técnicas de Cultura de TecidosRESUMO
Toll-like receptors (TLRs) play key roles in innate immune recognition of pathogen-associated molecular patterns of invading microbes. Among the 10 TLR family members identified in humans, TLR10 remains an orphan receptor without known agonist or function. TLR10 is a pseudogene in mice and mouse models are noninformative in this regard. Using influenza virus infection in primary human peripheral blood monocyte-derived macrophages and a human monocytic cell line, we now provide previously unidentified evidence that TLR10 plays a role in innate immune responses following viral infection. Influenza virus infection increased TLR10 expression and TLR10 contributed to innate immune sensing of viral infection leading to cytokine induction, including proinflammatory cytokines and interferons. TLR10 induction is more pronounced following infection with highly pathogenic avian influenza H5N1 virus compared with a low pathogenic H1N1 virus. Induction of TLR10 by virus infection requires active virus replication and de novo protein synthesis. Culture supernatants of virus-infected cells modestly up-regulate TLR10 expression in nonvirus-infected cells. Signaling via TLR10 was activated by the functional RNA-protein complex of influenza virus leading to robust induction of cytokine expression. Taken together, our findings identify TLR10 as an important innate immune sensor of viral infection and its role in innate immune defense and immunopathology following viral and bacterial pathogens deserves attention.
Assuntos
Imunidade Inata/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/imunologia , Receptor 10 Toll-Like/imunologia , Animais , Benzotiazóis , Western Blotting , Primers do DNA/genética , Diaminas , Cães , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Macrófagos , Células Madin Darby de Rim Canino , Camundongos , Compostos Orgânicos , Quinolinas , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 10 Toll-Like/metabolismoRESUMO
OBJECTIVES: The main function of influenza neuraminidase (NA) involves enzymatic cleavage of sialic acid from the surface of host cells resulting in the release of the newly produced virions from infected cells, as well as aiding the movement of virions through sialylated mucus present in the respiratory tract. However, there has previously been little information on the binding affinity of different forms of sialylated glycan with NA. Our objectives were then to investigate both sialic acid binding and cleavage of neuraminidase at an atomic resolution level. DESIGN: Nuclear magnetic resonance (NMR) spectroscopy was used to investigate pH and temperature effects on binding and cleavage as well as to interrogate the selectivity of human-like or avian-like receptors for influenza neuraminidase N1 derived from a range of different influenza virus strains including human seasonal H1N1, H1N1pdm09 and avian H5N1. RESULTS: We demonstrated that an acidic pH and physiological temperature are required for efficient NA enzymatic activity; however a change in the pH had a minimum effect on the NA-sialic acid binding affinity. Our data comparing α-2,3- and α-2,6-sialyllactose indicated that the variation in neuraminidase activity on different ligands correlated with a change in binding affinity. Epitope mapping of the sialylglycans interacting with NAs from different viral origin showed different binding profiles suggesting that different binding conformations were adopted. CONCLUSIONS: The data presented in this study demonstrated that physicochemical conditions (pH in particular) could affect the NA enzymatic activity with minor effect on ligand binding. NA cleavage specificity seemed to be associated with a difference in binding affinity to different ligands, suggesting a relationship between the two events. These findings have implications regarding the replication cycle of influenza infection in the host where different sialidase activities would influence penetration through the respiratory mucin barrier and the release of the newly generated virus from the infected cells.
Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Virus da Influenza A Subtipo H5N1/enzimologia , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Especificidade por Substrato , Proteínas Virais/metabolismo , Animais , Aves , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Espectroscopia de Ressonância Magnética , Neuraminidase/química , Ligação Proteica , Temperatura , Proteínas Virais/químicaAssuntos
Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/química , Orthomyxoviridae/enzimologia , Animais , Antivirais/farmacologia , Sítios de Ligação/efeitos dos fármacos , Humanos , Modelos Moleculares , Neuraminidase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Orthomyxoviridae/química , Infecções por Orthomyxoviridae/enzimologia , Oseltamivir/farmacologiaRESUMO
Pseudotyped viral particles are being used as safe surrogates to mimic the structure and surface of many viruses, including highly pathogenic viruses such as avian influenza H5N1, to investigate biological functions mediated by the envelope proteins derived from these viruses. The first part of this article evaluates and discusses the differences in the production and characterization of influenza pseudoparticles. The second part focuses on the applications that such a flexible tool can provide in modern influenza research, in particular in the fields of drug discovery, molecular biology and diagnosis.
Assuntos
Alphainfluenzavirus/fisiologia , Lentivirus/genética , Glicoproteínas de Membrana/metabolismo , Infecções por Orthomyxoviridae/diagnóstico , Proteínas Virais/metabolismo , Vírion/metabolismo , Animais , Doenças das Aves/diagnóstico , Doenças das Aves/virologia , Aves , Quimera/fisiologia , Descoberta de Drogas , Humanos , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Influenza Humana/diagnóstico , Influenza Humana/virologia , Lentivirus/metabolismo , Glicoproteínas de Membrana/genética , Biologia Molecular , Infecções por Orthomyxoviridae/virologia , Plasmídeos/fisiologia , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Transfecção , Vírion/genética , Vírion/isolamento & purificaçãoRESUMO
The minimal virus requirements for the generation of influenza virus-like particle (VLP) assembly and budding were reassessed. Using neuraminidase (NA) from the H5N1 and H1N1 subtypes, it was found that the expression of NA alone was sufficient to generate and release VLPs. Biochemical and functional characterization of the NA-containing VLPs demonstrated that they were morphologically similar to influenza virions. The NA oligomerization was comparable to that of the live virus, and the enzymic activity, whilst not required for the release of NA-VLPs, was preserved. Together, these findings indicate that NA plays a key role in virus budding and morphogenesis, and demonstrate that NA-VLPs represent a useful tool in influenza research.
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Neuraminidase/fisiologia , Proteínas Virais/fisiologia , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologia , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Humanos , Corpos de Inclusão Viral/genética , Corpos de Inclusão Viral/fisiologia , Corpos de Inclusão Viral/ultraestrutura , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/ultraestrutura , Virus da Influenza A Subtipo H5N1/ultraestrutura , Microscopia Eletrônica de Transmissão , Neuraminidase/genética , Transfecção , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/fisiologia , Proteínas Virais/genética , Montagem de Vírus/genética , Liberação de Vírus/genéticaRESUMO
To evaluate the effects of long term hypoxia exposure on fish spawning, mature common carp, Cyprinus carpio carpio (Linnaeus) were subjected to either normoxia (7.4+/-0.2 mgO(2)mg O(2) L(-1)) or hypoxia (1.0+/-0.2 mgO(2)O(2) L(-1)) for more than two months. Gonadosomatic index (GSI), and concentrations of serum luteinizing hormone (LH), testosterone (T), and estroldiol (E2) were measured and gonad histology examined. Hypoxia inhibits fish spawning even though the gonad and oocytes developed under hypoxia exposure. LH levels of female carp were significantly decreased upon chronic exposure to hypoxia, and the final oocyte maturation in hypoxic females was significantly retarded. The results indicated that hypoxia may inhibit fish spawning through LH-dependent final oocyte maturation. In addition, no courtship was observed in hypoxic males. In conclusion, hypoxia impairs fish ovulation and, therefore, spawning and reproduction. LH levels were reduced leading to a failure of oocyte maturation. This, along with a lack of courtship by males may be the major mechanisms involved in hypoxic inhibition of reproduction in carp.
Assuntos
Carpas/metabolismo , Hipóxia/metabolismo , Hormônio Luteinizante/sangue , Oogênese , Oviparidade , Oviposição , Animais , Corte , Estradiol/sangue , Feminino , Hipóxia/patologia , Hipóxia/fisiopatologia , Masculino , Ovário/metabolismo , Ovário/patologia , Comportamento Sexual Animal , Testículo/metabolismo , Testículo/patologia , Testosterona/sangue , Fatores de TempoAssuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/química , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Receptores de Superfície Celular/química , Vírus da Influenza A/classificação , Tamanho da Partícula , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Erythropoietin (EPO) is a glycoprotein hormone that regulates the proliferation and differentiation of erythroid progenitor cells in mammals. Although EPO has been identified in fish, the specific function and effects of hypoxia have not been investigated previously. In this study, we have demonstrated a relationship between increases in renal EPO levels and decreases in spleen EPO levels and spleen-somatic index (SSI), with increases in haemoglobin (Hb) concentration in the blood during hypoxia exposure in rainbow trout. Splenic contraction and the subsequent red blood cell release accounts for the initial increase in Hb concentration in the blood, whereas EPO action probably accounts for the later increases in hemoglobin concentration in the blood. Our data indicate that fish and mammalian erythropoietic systems are similar in response to hypoxia, in that erythropoiesis in fish is influenced by EPO.