Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion
ABSTRACT West Nile virus (WNV) transmission by transfusion was documented in 2002. Approximately 80 percent of WNV infections are asymptomatic and 1 percent develop severe neurological illness. In animals, Langerhans-dendritic cells support initial viral replication, followed by replication in lymphoid tissues and dissemination to organs and possibly to the CNS. The cellular tropism of WNV infection after transfusion and the particular human blood cells that sustain viral replication remain largely unknown. Whether primary monocyte-derived macrophages (MDMs) support WNV infection-replication and produce infectious virions, with an in vitro system, was investigated.
Elutriated monocytes (CD33+/CD14+) from suitable blood donors were cultured in the presence of macrophage-colony-stimulating factor, infected with WNV-NY99 at different time points, washed, and cultivated for up to 47 days. Supernatants were tested for WNV replication by TaqMan reverse transcription-polymerase chain reaction (RT-PCR), with primers for the envelope and/or 3'NC regions, and by cDNA-PCR to detect WNV minus-strand RNA and for the presence of functional virions by infectivity assays in Vero cells.
RT-PCR TaqMan of supernatants demonstrated productive infection of MDMs. Viral load reached 2 to 5 log above baseline in 3 to 6 days and then declined, with detectable viral replication persisting for up to 47 days. WNV minus-strand RNA was detected in Day 4 cultures, indicating active viral replication. Infected MDM cultures showed no cytopathic changes. Supernatants that were TaqMan-positive for the presence of WNV-infected Vero cells and produced cytopathic effects within 3 to 5 days of culture.
The susceptibility of monocytes-macrophages to productive infection in vitro is compatible with a potential role in initial WNV replication and propagation after transmission by transfusion.
SourceAvailable from: Takashi Onodera[Show abstract] [Hide abstract]
ABSTRACT: Two monocytic cell lines, temporary named HsMo (horse monocyte) and MsMo (mouse monocyte) were established from blood samples of horse and Balb/C mouse according to the culture method previously informed (Kadoi, 2011). Tests were performed to certify the viral susceptibility of these cells in comparison with that of Vero cells for West Nile virus (WNV). Two strains of WNV adapted to grow in Vero cells were titrated for infectivity in the monocytic cells and Vero cells in a routine microplate assay system. The highest titer was obtained in the assay system with HsMo cells followed by the system with MsMo cells. These monocytic cells were sensitive enough for detecting WNV, however, WNV infected monocytes produced a strong acidic substance in which the acidity is much higher grade than that produced in Vero cells. In microplate culture the color of medium changes from redden neutral color to yellow indicated by phenol red dye included in cell culture medium, in parallel with CPE occurrence. This is a color test for WNV assay in monocytic cells Keywords: Horse monocytes, mouse monocytes, WNV susceptibility. INTRDUCTION The invasion of West Nile virus (WNV) became a worldwide serious problem since the causative pathogenic virus has readily spread in many countries, Africa, the Middle East, Europe, USA, USSR, and Asia. (Dauphin et al., 2004; Zeller and Schuffenecker, 2004; Hayes et al., 2005). WNV is an arbovirus transmitted by various mosquito species (Anderson et al., 1999). This is the main reason for the elimination of the disease once introduced to virgin area. Several animal species are known to be susceptible hosts (Lanciotti et al., 1999; Weingartle et al., 2004) and birds are suspected to be amplifying host (Anderson et al., 1999; Kramer and Bernard, 2001; McLean et al., 2001; Weingartle et al., 2004). The virus was isolated from samples of humans followed by horses in the beginning of the history. However, it was now understood that the virus is widely distributed in animal world since the virus was isolated not only clinically affected animals, but was also isolated from asymptomatic animals as camel and grass mouse,
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ABSTRACT: Previous studies have shown that an attenuated West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant induces stronger innate and adaptive immune responses than wild-type WNV in mice, which has important applications to vaccine development. To investigate the mechanism of immunogenicity, we characterized WNV NS4B-P38G mutant infection in two human cell lines-THP-1 cells and THP-1 macrophages. Although the NS4B-P38G mutant produced more viral RNA than the parental WNV NY99 in both cell types, there was no detectable infectious virus in the supernatant of either cell type. Nonetheless, the attenuated mutant boosted higher innate cytokine responses than virulent parental WNV NY99 in these cells. The NS4B-P38G mutant infection of THP-1 cells led to more diverse and robust innate cytokine responses than that seen in THP-1 macrophages, which were mediated by toll-like receptor (TLR)7 and retinoic acid-inducible gene 1(RIG-I) signaling pathways. Overall, these results suggest that a defective viral life cycle during NS4B-P38G mutant infection in human monocytic and macrophage cells leads to more potent cell intrinsic innate cytokine responses. Copyright © 2014. Published by Elsevier Ltd.Vaccine 01/2015; 33(7). DOI:10.1016/j.vaccine.2014.12.056 · 3.49 Impact Factor
01/2013, Degree: PhD, Supervisor: Hans Nauwynck