RESUMO
Avian A(H7N9) infections in humans have been reported in China since 2013 and are of public health concern due to their severity and pandemic potential. Oseltamivir and peramivir are neuraminidase inhibitors (NAIs) routinely used for the treatment of A(H7N9) infections, but variants with reduced sensitivity to these drugs can emerge in patients during treatment. Zanamivir and laninamivir are NAIs that are used less frequently. Herein, we performed in vitro serial passaging experiments with recombinant viruses, containing the neuraminidase (NA) from influenza A/Anhui/1/13 (H7N9) virus, in the presence of each NAI, to determine whether variants with reduced sensitivity would emerge. NA substitutions were characterized for their effect on the NA enzymatic activity and surface expression of the A/Anhui/1/13 (Anhui/1) NA, as well as NAs originating from contemporary A(H7N9) viruses of the Yangtze River Delta and Pearl River Delta lineages. In vitro passage in the presence of oseltamivir, peramivir and laninamivir selected for substitutions associated with reduced sensitivity (E119D, R292K and R152K), whereas passage in the presence of zanamivir did not select for any viruses with reduced sensitivity. All the NA substitutions significantly reduced activity, but not the expression of the Anhui/1 NA. In contemporary N9 NAs, all substitutions tested significantly reduced NA enzyme function in the Yangtze River lineage background, but not in the Pearl River Delta lineage background. Overall, these findings suggest that zanamivir may be less likely than the other NAIs to select for resistance in A(H7N9) viruses and that the impact of substitutions that reduce NAI susceptibility or enzyme function may be less in A(H7N9) viruses from the Pearl River lineage.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Ácidos Carbocíclicos , Substituição de Aminoácidos , Animais , Ciclopentanos/farmacologia , Cães , Farmacorresistência Viral/efeitos dos fármacos , Guanidinas/farmacologia , Células HEK293 , Humanos , Subtipo H7N9 do Vírus da Influenza A/classificação , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Subtipo H7N9 do Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Neuraminidase/metabolismo , Oseltamivir/farmacologia , Piranos , Inoculações Seriadas , Ácidos Siálicos , Especificidade da Espécie , Proteínas Virais/metabolismo , Zanamivir/análogos & derivados , Zanamivir/farmacologiaRESUMO
Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.
Assuntos
Inibidores Enzimáticos/farmacologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/genética , Neuraminidase/antagonistas & inibidores , Substituição de Aminoácidos/efeitos dos fármacos , Substituição de Aminoácidos/genética , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Farmacorresistência Viral/genética , Feminino , Furões , Células HEK293 , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Masculino , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Influenza viruses A/PR/8/34 (PR8; H1N1), A/Aichi/68 X-31 (HKx31; H3N2), and A/Beijing/89 X-109 (BJx109; H3N2) show marked differences in their ability to infect murine macrophages, including resident alveolar and peritoneal macrophages as well as the macrophage-derived cell line J774. The hierarchy in infectivity of the viruses (PR8 < HKx31 < BJx109) resembles that of their reactivity with mannose-binding lectins of the collectin family. Since the macrophage mannose receptor recognizes the same spectrum of monosaccharides as the collectins do, we investigated the possible involvement of this receptor in infection of macrophages by influenza virus. In competitive binding studies, the binding of (125)I-labeled mannosylated bovine serum albumin to macrophages was inhibited by the purified hemagglutinin and neuraminidase (HANA) glycoproteins of influenza virus but not by HANA that had been treated with periodate to oxidize its oligosaccharide side chains. The inhibitory activity of HANA from the three strains of virus differed markedly and correlated with the infectivity of each virus for macrophages. Infection of macrophages, but not MDCK cells, by influenza virus was inhibited by yeast mannan. A variant line of J774 cells, J774E, which expresses elevated levels of the mannose receptor, was more readily infected than J774, and the sensitivity of J774E cells to infection was greatly reduced by culture in the presence of D-mannose, which down-modulated mannose receptor expression. Together, the data implicate the mannose receptor as a major endocytic receptor in the infectious entry of influenza virus, and perhaps other enveloped viruses, into murine macrophages.
Assuntos
Vírus da Influenza A/metabolismo , Lectinas Tipo C , Macrófagos Alveolares/virologia , Macrófagos Peritoneais/virologia , Lectinas de Ligação a Manose , Receptores de Superfície Celular/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A/fisiologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Mananas/metabolismo , Receptor de Manose , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismoRESUMO
The antiviral activity against rotaviruses of three bovine collectins, conglutinin, collectin-43 (CL-43) and bovine SP-D, was examined. As shown by ELISA and Western blot, all three collectins bound to the Nebraska calf diarrhoea virus bovine strain of rotavirus, and specifically to the VP7 glycoprotein. Inhibition by mannose or EDTA confirmed that binding was mediated through the lectin domains of the collectins. Binding resulted in haemagglutination inhibition and neutralization of rotavirus infectivity, CL-43 displaying the highest activity in both types of assay. In contrast, conglutinin was the most potent of the three collectins against influenza virus A/HKx31. Neutralization of rotaviruses by the lectins was dependent on glycosylation of VP7. Furthermore, rotaviruses adapted to growth in Madin-Darby bovine kidney cells, and thus bearing carbohydrate of bovine origin, remained sensitive to neutralization, although slightly less so than virus stocks propagated in the monkey kidney cell line MA104. These findings provide the first description of antiviral activity of collectins against a non-enveloped virus and may indicate a potential role for collectins in host defence against bovine rotavirus infection.
Assuntos
Antígenos Virais , Antivirais/farmacologia , Proteínas do Capsídeo , Colectinas , Glicoproteínas/farmacologia , Lectinas/farmacologia , Surfactantes Pulmonares/farmacologia , Rotavirus/efeitos dos fármacos , Soroglobulinas/farmacologia , Animais , Antivirais/metabolismo , Sítios de Ligação , Capsídeo/química , Capsídeo/imunologia , Capsídeo/metabolismo , Bovinos , Linhagem Celular , Ácido Edético/farmacologia , Glicoproteínas/metabolismo , Glicosilação , Testes de Inibição da Hemaglutinação , Vírus da Influenza A/imunologia , Vírus da Influenza A/metabolismo , Lectinas/metabolismo , Manose/farmacologia , Testes de Neutralização , Proteína D Associada a Surfactante Pulmonar , Surfactantes Pulmonares/metabolismo , Rotavirus/imunologia , Rotavirus/metabolismo , Soroglobulinas/metabolismoRESUMO
The influence of diabetes on susceptibility to influenza virus infection was examined in a mouse model in which RIP-Kb transgenic mice and their nontransgenic littermates were used as the diabetic and nondiabetic hosts, respectively. Influenza virus A/Phil/82 (H3N2) grew to significantly higher titers in the lungs of diabetic than nondiabetic mice. The extent of viral replication in the lungs was proportional to blood glucose levels in the mice at the time of infection, and the enhanced susceptibility of diabetic mice was reversed with insulin. Growth of A/HKx31 (H3N2) virus was also enhanced in diabetic mice, whereas the highly virulent strain A/PR/8/34 (H1N1) showed no difference in virus yields in diabetic and nondiabetic mice, even with low inocula. A/Phil/82 and A/HKx31 are sensitive to neutralization in vitro by the pulmonary collectin surfactant protein D (SP-D), whereas A/PR/8/34 is essentially resistant. Glucose is a ligand for SP-D, and neutralization of A/Phil/82 virus by SP-D was abolished in the presence of glucose at levels commonly found in diabetic mice. These findings suggest that in mice, and perhaps in humans, diabetes predisposes to influenza virus infection through compromise of collectin-mediated host defense of the lung by glucose.
Assuntos
Diabetes Mellitus Experimental/complicações , Glicoproteínas/metabolismo , Vírus da Influenza A/metabolismo , Influenza Humana/complicações , Surfactantes Pulmonares/metabolismo , Animais , Glicemia , Linhagem Celular , Suscetibilidade a Doenças , Cães , Feminino , Glicoproteínas/imunologia , Glicoproteínas/farmacologia , Humanos , Vírus da Influenza A/crescimento & desenvolvimento , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína D Associada a Surfactante Pulmonar , Surfactantes Pulmonares/imunologia , Surfactantes Pulmonares/farmacologia , Ratos , Fatores de RiscoRESUMO
Collagenous lectins (collectins) present in mammalian serum and pulmonary fluids bind to influenza virus and display antiviral activity in vitro, but their role in vivo has yet to be determined. We have used early and late isolates of H3N2 subtype influenza viruses that differ in their degree of glycosylation to examine the relationship between sensitivity to murine serum and pulmonary lectins in vitro and the ability of a virus to replicate in the respiratory tract of mice. A marked inverse correlation was found between these two parameters. Early H3 isolates (1968 to 1972) bear 7 potential glycosylation sites on hemagglutinin (HA), whereas later strains carry 9 or 10. Late isolates were shown to be much more sensitive than early strains to neutralization by the mouse serum mannose-binding lectin (MBL) and rat lung surfactant protein D (SP-D) and bound greater levels of these lectins in enzyme-linked immunosorbent assays and Western blot analyses. They also replicated very poorly in mouse lungs compared to the earlier strains. Growth in the lungs was greatly enhanced, however, if saccharide inhibitors of the collectins were included in the virus inoculum. The level of SP-D in bronchoalveolar lavage fluids increased on influenza virus infection. MBL was absent from lavage fluids of normal mice but could be detected in fluids from mice 3 days after infection with the virulent strain A/PR/8/34 (H1N1). The results implicate SP-D and possibly MBL as important components of the innate defense of the respiratory tract against influenza virus and indicate that the degree or pattern of glycosylation of a virus can be an important factor in its virulence.
Assuntos
Proteínas de Transporte/fisiologia , Glicoproteínas/fisiologia , Lectinas/metabolismo , Pulmão/virologia , Infecções por Orthomyxoviridae/fisiopatologia , Surfactantes Pulmonares/fisiologia , Animais , Colectinas , Feminino , Glicoproteínas/química , Glicosilação , Lectinas de Ligação a Manose , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/imunologia , Proteína D Associada a Surfactante Pulmonar , Ratos , Sistema Respiratório/virologia , Relação Estrutura-Atividade , Traqueia/virologia , Replicação ViralRESUMO
The H3N2 influenza virus A/Philippines/82 (Phil82) and its bovine serum-resistant mutant, Phil82/BS, were used to investigate factors that influence virulence of influenza virus for mice. Phil82/BS, which lacks the high-mannose oligosaccharide at residue 165 of the hemagglutinin (HA) molecule, was found to replicate to a much higher titer in mouse lung than the parent Phil82, and had acquired lethality for mice. Further adaptation of Phil82/BS by sequential lung passage in mice yielded a strain of greater virulence, Phil82/BS/ML 10, in which a change at residue 246 of HA resulted in loss of a second potential glycosylation site. Phil82 is highly sensitive to neutralization in vitro by murine serum- and lung-associated mannose-binding lectins (collectins). Characterization of the two mutant viruses indicated that resistance to murine collectins can account for the enhanced virulence of Phil82/BS but not for the further increase in virulence of Phil82/BS/ML10. Evidence is presented that residue 246 is not in fact glycosylated in Phil82/BS HA, nor presumably in the parent Phil82 virus. The HA molecule of Phil82/BS/ML10 displayed functional differences from Phil82/BS, including a change in the optimum pH of fusion and a minor change in receptor-binding specificity, which may allow improved efficiency of replication in the mouse lung.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/genética , Animais , Bovinos , Feminino , Vírus da Influenza A/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Virulência/genéticaRESUMO
The mechanism of lysis of influenza virus-infected BHK-21 cells by guinea pig serum (GPS) was investigated. Lysis was shown to involve activation of the classical complement pathway and was dependent on the presence of a mannose-binding lectin in GPS. FACS analysis demonstrated Ca(2+)-dependent binding of the lectin to influenza virus-infected, but not uninfected, cells. Cells infected with mutant strains of virus lacking a particular high-mannose oligosaccharide at the tip of the hemagglutinin molecule showed reduced binding of the lectin and were correspondingly less sensitive to lysis by GPS than cells infected with the parent viruses. The degree or pattern of glycosylation of influenza viruses thus influences susceptibility to this mechanism of viral clearance. By interfering with the infectious process, lectin-dependent complement-mediated lysis of infected cells may be an important component of innate immunity to influenza and other enveloped viruses.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Vírus da Influenza A/imunologia , Lectinas/metabolismo , Manose/metabolismo , Infecções por Orthomyxoviridae/imunologia , Animais , Células Cultivadas , Via Clássica do Complemento , Cricetinae , Testes Imunológicos de Citotoxicidade , CobaiasRESUMO
The nature of the beta inhibitor in guinea-pig serum and its mechanism of neutralization of influenza virus have been investigated. This inhibitor was shown to be a mannose-binding lectin serologically related to human serum mannose-binding protein. Ca(2+)-dependent binding of the guinea-pig lectin to influenza virus or to mannan could be detected with polyclonal or monoclonal antibodies against human mannose-binding protein in an ELISA. Furthermore, the monoclonal antibody inhibited both the haemagglutination-inhibiting and virus-neutralizing activities of the guinea-pig lectin. The lectin was active against influenza viruses of both type A and type B. In haemagglutination inhibition it acts independently of complement, apparently by sterically hindering access to the receptor-binding site on the viral haemagglutinin through binding of the lectin to carbohydrate side-chains in the vicinity of this site. Neutralization by the lectin, however, was shown to require activation of the classical complement pathway. To our knowledge, the neutralization of influenza virus by a serum lectin plus complement represents a previously unrecognized mechanism of complement-dependent viral inactivation that may be important in first-line host defence against a variety of enveloped viruses.