RESUMEN
Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using the QIAamp viral RNA minikit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe, and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection.
Asunto(s)
Desinfección/métodos , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre Hemorrágica Ebola/virología , Sistemas de Atención de Punto , ARN Viral/aislamiento & purificación , Manejo de Especímenes/métodos , Inactivación de Virus , Humanos , Temperatura , Factores de TiempoRESUMEN
Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.
Asunto(s)
Apoptosis/inmunología , Infecciones por Hantavirus/inmunología , Células Endoteliales de la Vena Umbilical Humana/virología , Células Asesinas Naturales/virología , Orthohantavirus/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Caspasa 3/metabolismo , Inhibidores de Caspasas , Degranulación de la Célula , Células Cultivadas , Técnicas de Cocultivo , Pruebas Inmunológicas de Citotoxicidad , Granzimas/antagonistas & inhibidores , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Datos de Secuencia MolecularRESUMEN
Hantaviruses, the causative agents of two emerging diseases, are negative-stranded RNA viruses with a tripartite genome. We isolated two substrains from a parental strain of Puumala hantavirus (PUUV-Pa), PUUV-small (PUUV-Sm) and PUUV-large (PUUV-La), named after their focus size when titrated. The two isolates were sequenced; this revealed differences at two positions in the nucleocapsid protein and two positions in the RNA-dependent RNA polymerase, but the glycoproteins were identical. We also detected a 43-nucleotide deletion in the PUUV-La S-segment 5' noncoding region covering a predicted hairpin loop structure that was found to be conserved among all hantaviruses with members of the rodent subfamily Arvicolinae as their hosts. Stocks of PUUV-La showed a lower ratio of viral RNA to infectious particles than stocks of PUUV-Sm and PUUV-Pa, indicating that PUUV-La replicated more efficiently in alpha/beta interferon (IFN-α/ß)-defective Vero E6 cells. In Vero E6 cells, PUUV-La replicated to higher titers and PUUV-Sm replicated to lower titers than PUUV-Pa. In contrast, in IFN-competent MRC-5 cells, PUUV-La and PUUV-Sm replicated to similar levels, while PUUV-Pa progeny virus production was strongly inhibited. The different isolates clearly differed in their potential to induce innate immune responses in MRC-5 cells. PUUV-Pa caused stronger induction of IFN-ß, ISG56, and MxA than PUUV-La and PUUV-Sm, while PUUV-Sm caused stronger MxA and ISG56 induction than PUUV-La. These data demonstrate that the phenotypes of isolated hantavirus substrains can have substantial differences compared to each other and to the parental strain. Importantly, this implies that the reported differences in phenotypes for hantaviruses might depend more on chance due to spontaneous mutations during passage than inherited true differences between hantaviruses.
Asunto(s)
Células Epiteliales/virología , Fibroblastos/virología , Riñón/virología , Pulmón/virología , Virus Puumala/clasificación , Virus Puumala/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Riñón/citología , Pulmón/citología , Datos de Secuencia Molecular , Mutación , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Fenotipo , Virus Puumala/genética , Virus Puumala/inmunología , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Células Vero , Proteínas Virales/química , Proteínas Virales/genética , Replicación ViralRESUMEN
Type III interferons ([IFNs] IFN-lambda and interleukin-28 and -29 [IL-28/29]) are recently recognized cytokines with innate antiviral effects similar to those of type I IFNs (IFN-alpha/beta). Like IFN-alpha/beta, IFN-lambda-expression can be induced by viruses, and it is believed that type I and III IFNs are regulated in the same manner. Hantaviruses are weak IFN-alpha/beta inducers and have surprisingly been shown to activate IFN-alpha/beta-independent IFN-stimulated gene (ISG) expression. Here, we show that in Hantaan virus (HTNV)-infected human epithelial A549 cells, induction of IFN-lambda1 preceded induction of MxA and IFN-beta by 12 and 24 h, respectively, and IFN-alpha was not induced at all. Furthermore, induction of IFN-lambda1 and MxA was observed in HTNV-infected African green monkey epithelial Vero E6 cells, a cell line that cannot produce type I IFNs, clearly showing that HTNV can induce IFN-lambda1 and ISGs in the complete absence of IFN-alpha/beta. In HTNV-infected human fibroblast MRC-5 cells, which lack the IFN-lambda receptor, induction of MxA coincided in time with IFN-beta-induction. UV-inactivated HTNV did not induce any IFNs or MxA in any cell line, showing that activation of IFN-lambda1 is dependent on replicating virus. Induction of both IFN-beta and IFN-lambda1 in A549 cells after poly(I:C)-stimulation was strongly inhibited in HTNV-infected cells, suggesting that HTNV can inhibit signaling pathways used to simultaneously activate types I and III IFNs. In conclusion, we show that HTNV can cause type I IFN-independent IFN-lambda1 induction and IFN-lambda1-specific ISG induction. Importantly, the results suggest the existence of specific signaling pathways that induce IFN-lambda1 without simultaneous type I IFN induction during virus infection.
Asunto(s)
Virus Hantaan/inmunología , Interleucinas/biosíntesis , Animales , Línea Celular , Chlorocebus aethiops , Células Epiteliales/inmunología , Células Epiteliales/virología , Humanos , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Interferón beta/inmunología , Interferón beta/metabolismo , Interferones , Interleucinas/inmunología , Transducción de SeñalRESUMEN
BACKGROUND: Hantaviruses cause two severe and often fatal human diseases: haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Presently, there is no effective prevention available for HFRS or HPS. Here, we studied the effect of passive immunization on the course of infection in cynomolgus macaques challenged with wild-type Puumala hantavirus (PUUV-wt). METHODS: A pool of serum drawn from previously PUUV-wt-infected monkeys was used for immunization; a pool of serum from the same monkeys that was obtained before infection was used as a control. Immunizations were administered 3 days before and 15 days after challenge with PUUV-wt. After challenge, monkeys were sampled once a week and analysed for PUUV-infection markers. RESULTS: All three monkeys treated with non-immune serum became positive for PUUV RNA in plasma and showed PUUV nucleocapsid-specific immunoglobin M (IgM) responses after challenge. In contrast, no PUUV RNA or anti-PUUV-specific IgM response was detected in the three passively immunized monkeys. As seen in PUUV-infected humans, the control monkeys showed a marked decrease in the amount of platelets and increased levels of creatinine, interleukin (1L)-6, IL-10, and tumour necrosis factor (TNF) after inoculation. In contrast, no marked changes in the amount of platelets were observed in the immunized monkeys and they did not show increased levels of creatinine, IL-6, IL-10 and TNF after virus challenge. CONCLUSION: The results show that passive immunization in monkeys, using serum from previously hantavirus-infected monkeys, can induce sterile protection and protect against pathogenesis. Convalescent-phase antibodies may represent a potential therapy that can induce immediate protection against HFRS and HPS.
Asunto(s)
Fiebre Hemorrágica con Síndrome Renal/inmunología , Inmunización Pasiva , Macaca fascicularis/inmunología , Macaca fascicularis/virología , Virus Puumala/inmunología , Animales , Anticuerpos Antivirales/sangre , Proteína C-Reactiva/metabolismo , Creatinina/sangre , Citocinas/sangre , Óxido Nítrico/sangre , Factores de Tiempo , Viremia/inmunologíaRESUMEN
The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-ß and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-ß or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-ß but not Mx2, while TBEV induced both IFN-ß and Mx2. In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.
Asunto(s)
Arvicolinae/virología , Reservorios de Enfermedades/virología , Modelos Biológicos , Fenómenos Fisiológicos de los Virus , Animales , Arvicolinae/genética , Línea Celular , Virus de la Viruela Vacuna/efectos de los fármacos , Virus de la Viruela Vacuna/inmunología , Embrión de Mamíferos/citología , Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/virología , Humanos , Inmunidad Innata/efectos de los fármacos , Interferón beta/farmacología , Parechovirus/efectos de los fármacos , Parechovirus/inmunología , Virus Puumala/efectos de los fármacos , Virus Puumala/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Fenómenos Fisiológicos de los Virus/efectos de los fármacos , Virus/efectos de los fármacosRESUMEN
Natural killer (NK) cells are known to mount a rapid response to several virus infections. In experimental models of acute viral infection, this response has been characterized by prompt NK cell activation and expansion followed by rapid contraction. In contrast to experimental model systems, much less is known about NK cell responses to acute viral infections in humans. We demonstrate that NK cells can rapidly expand and persist at highly elevated levels for >60 d after human hantavirus infection. A large part of the expanding NK cells expressed the activating receptor NKG2C and were functional in terms of expressing a licensing inhibitory killer cell immunoglobulin-like receptor (KIR) and ability to respond to target cell stimulation. These results demonstrate that NK cells can expand and remain elevated in numbers for a prolonged period of time in humans after a virus infection. In time, this response extends far beyond what is considered normal for an innate immune response.
Asunto(s)
Infecciones por Hantavirus/inmunología , Células Asesinas Naturales/inmunología , Proliferación Celular , Células Endoteliales/inmunología , Humanos , Células Asesinas Naturales/citología , Ligandos , Subfamília C de Receptores Similares a Lectina de Células NK/inmunología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), are known to be sensitive to nitric oxide (NO) and to pretreatment with type I and II interferons (alpha interferon [IFN-alpha]/IFN-beta and IFN-gamma, respectively). Elevated serum levels of NO and IFN-gamma have been observed in HFRS patients, but little is known regarding the systemic levels of other IFNs and the possible effects of hantaviruses on innate antiviral immune responses. In Puumala virus-infected HFRS patients (n = 18), we report that the levels of IFN-alpha and IFN-beta are similar, whereas the level of IFN-lambda (type III IFN) is significantly decreased, during acute (day of hospitalization) compared to the convalescent phase. The possible antiviral effects of IFN-lambda on the prototypic hantavirus Hantaan virus (HTNV) replication was then investigated. Pretreatment of A549 cells with IFN-lambda alone inhibited HTNV replication, and IFN-lambda combined with IFN-gamma induced additive antiviral effects. We then studied the effect of postinfection treatment with IFNs. Interestingly, an already-established HTNV infection was insensitive to subsequent IFN-alpha, -beta, -gamma, and -lambda stimulation, and HTNV-infected cells produced less NO compared to noninfected cells when stimulated with IFN-gamma and IL-1beta. Furthermore, less phosphorylated STAT1 after IFN treatment was observed in the nuclei of infected cells than in those of noninfected cells. The results suggest that hantavirus can interfere with the activation of antiviral innate immune responses in patients and inhibit the antiviral effects of all IFNs. We believe that future studies addressing the mechanisms by which hantaviruses interfere with the activation and shaping of immune responses may bring more knowledge regarding HFRS and HCPS pathogenesis.
Asunto(s)
Citocinas/sangre , Síndrome Pulmonar por Hantavirus/inmunología , Interferones/farmacología , Interleucinas/sangre , Orthohantavirus/efectos de los fármacos , Enfermedad Cardiopulmonar/inmunología , Enfermedad Cardiopulmonar/virología , Células Cultivadas , Citocinas/farmacología , Orthohantavirus/inmunología , Humanos , Interferón-alfa/sangre , Interferón-alfa/farmacología , Interferón gamma/sangre , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Interleucinas/farmacología , Óxido Nítrico/metabolismo , Fosforilación , Factor de Transcripción STAT1/metabolismo , Síndrome , Replicación Viral/efectos de los fármacosRESUMEN
Hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome are two diseases caused by hantaviruses. Capillary leakage is a hallmark of hantavirus infection. Pathogenic hantaviruses are not cytotoxic, but elevated levels of serum lactate dehydrogenase (LDH), indicative of cellular damage, are observed in patients. We report increased levels of serum perforin, granzyme B, and the epithelial cell apoptosis marker caspase-cleaved cytokeratin-18 during Puumala hantavirus infection. Significant correlation was observed between the levels of LDH and perforin and the levels of LDH and caspase-cleaved cytokeratin-18, suggesting that tissue damage is due to an immune reaction and that epithelial apoptosis contributed significantly to the damage.
Asunto(s)
Apoptosis , Células Epiteliales/patología , Fiebre Hemorrágica con Síndrome Renal/patología , Glicoproteínas de Membrana/sangre , Virus Puumala , Caspasas/metabolismo , Membrana Celular/patología , Membrana Celular/virología , Células Epiteliales/química , Células Epiteliales/virología , Granzimas , Fiebre Hemorrágica con Síndrome Renal/sangre , Humanos , Queratinas/análisis , L-Lactato Deshidrogenasa/análisis , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/sangreRESUMEN
Reactive nitrogen intermediates (RNI), like nitric oxide (NO) and peroxynitrite, have antiviral effects against certain viruses. Hantaviruses, like other members of the Bunyaviridae family, have previously not been shown to be sensitive to RNI. In this study, we compared the effects of NO and peroxynitrite on hantavirus replication and free mature virions in vitro, and of inducible nitric oxide synthase (iNOS) in hantavirus-infected suckling mice. The NO-generating compound S-nitroso-N-acetylpenicillamine (SNAP), as well as cytokine-induced NO, strongly inhibited hantavirus replication in Vero E6 cells, while pretreatment of free virions with SNAP only had a limited effect on their viability. In contrast, 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor, inhibited virus replication only to a very low extent in vitro, but pretreatment of virus with SIN-1 led to a considerably lowered viability of the virions. Infections of various human cell types per se did not induce NO production. The viral titers in iNOS(-/-) mice were higher compared to the controls, suggesting that NO inhibits hantavirus replication in vivo. In conclusion, we show that NO has strong antiviral effects on hantavirus replication, and peryoxynitrite on mature free virions, suggesting that different RNI can have different effects on various parts of the replication cycle for the same virus.